Retinoic acid receptor antagonists as chaperone-mediated autophagy modulators and uses thereof

ABSTRACT

Compounds, compositions and methods are provided for selectively activating chaperone-mediated autophagy (CMA), protecting cells from oxidative stress, proteotoxicity and lipotoxicity, and/or antagonizing activity of retinoic acid receptor alpha (RARα) in subjects in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/298,280, filed Oct. 20, 2016, which is a divisional of U.S. patentapplication Ser. No. 14/566,762, filed Dec. 11, 2014, now U.S. Pat. No.9,512,092 B2, issued Dec. 6, 2016, which claims the benefit of U.S.Provisional Patent Application No. 61/915,063, filed Dec. 12, 2013, thecontents of which are incorporated herein by reference in theirentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersAG021904, AG031782, HL095929 and AA020630 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to retinoic acid receptor antagonists aschaperone-mediated autophagy (CMA) modulators and uses thereof fortreatment of diseases and disorders such as neurodegenerative diseasesand diabetes and other diseases that could benefit by protecting cellsfrom oxidative stress, proteotoxicity, and lipotoxicity.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to insuperscripts. Citations for these references may be found at the end ofthe specification immediately preceding the claims. The disclosures ofthese publications are hereby incorporated by reference in theirentireties into the subject application to more fully describe the artto which the subject application pertains.

Autophagy is the process by which intracellular components undergodegradation in lysosomes^(1,2), contributing in this way to themaintenance of cellular homeostasis and to cellular quality control. Inaddition, autophagy is upregulated as a mechanism of cellular defenseagainst aggressors or to allow cellular adaptation to changingenvironmental conditions³. Alterations of the autophagic process havebeen described in multiple pathological conditions and underlie thepathogenesis of severe human diseases such as neurodegeneration, cancerand metabolic disorders^(1,4).

The best characterized autophagic pathways are macroautophagy andchaperone-mediated autophagy (CMA)¹. The distinctive characteristic ofCMA is that the specific subset of cytosolic proteins degraded by thispathway are directly translocated across the lysosomal membrane into thelysosomal lumen for degradation⁵. Substrates for this pathway all bearin their amino acid sequence a targeting motif⁶ that once recognized bythe cytosolic chaperone hsc70, mediates substrate delivery to thesurface of lysosomes⁷. Once there, substrates bind to thelysosome-associated membrane protein type 2A (LAMP-2A) and promote itsmultimerization into a high molecular weight complex, required forsubstrate translocation⁸. A variant of hsc70 resident in the lysosomallumen assists substrates to achieve complete translocation insidelysosomes.

CMA is maximally activated in response to stressors such as prolongednutritional deprivation, oxidative stress, hypoxia or exposure todifferent toxic compounds⁵. Malfunctioning of CMA has been described inneurodegenerative conditions such as familial forms of Parkinson'sdisease^(9,10) and certain tauopathies¹¹, in metabolic disorders such asdiabetes¹² and in different lysosomal storage disorders³¹. Furthermore,the gradual decline in the activity of this pathway with age has beenproposed to act as an aggravating factor in different age-relateddisorders¹⁴. In fact, if the reduction in CMA activity with age isprevented in vivo, through genetic manipulation in a mouse model,cellular homeostasis and organ function can be preserved until late inlife¹⁵. These findings, along with the growing number of connectionsbetween CMA and human diseases, justify the growing interest indeveloping efficient chemical modulators of this autophagic pathway.

Chemical compounds shown to have an effect on CMA until now lackselectivity for this pathway¹⁶. For example, inhibition of proteinsynthesis or of lysosomal proteases results in reduced CMA degradation,but it also affects many other intracellular processes¹⁶. Inhibition ofglucose-6-phosphate dehydrogenase or of the cytosolic chaperone hsp90lead to higher CMA activity in some cell types but not in others¹⁶; andin fact, later studies demonstrated that the effect was not direct but aconsequence of compensatory upregulation of other CMA component⁸. One ofthe limitations for the future development of CMA modulators has beenthe lack of information on the cellular signaling mechanisms thatactivate this pathway.

Retinoic acid receptors (RARs) act as transcriptional activators andrepressors of a broad subset of genes, contributing thus to modulatecellular processes in which CMA has also been involved, such asdifferentiation, proliferation and control of cellular homeostasis¹⁷.Furthermore, RAR loss or aberrant function has been described in manyoncogenic processes, where CMA upregulation is a common feature requiredto sustain cancer cell growth¹⁸. The three types of RARs identified inmammals, RARα, RARβ and RARγ, are coded by three different genes¹⁷. Incontrast to the complex tissue-dependent expression of RARβ and RARγ,RARα is ubiquitously expressed.

RAR are attractive druggable targets because their natural substrates,all-trans-retinoic acid (ATRA) and similar retinoids have been wellcharacterized¹⁹. Their efficient trafficking across lipid bilayers, dueto their small size and hydrophobic character²⁰, along with the growingunderstanding of the chemical modifications that the different regionsof retinoid derivatives can undergo intracellularly^(21,22) explains whyATRA by-products and derivatives are being already explored fortherapeutic purposes.

The present invention address the need for compounds that affectretinoic acid receptor (RAR) signaling and CMA activity and the use ofthese compounds in treatment of diseases and conditions associated withloss of CMA activity.

SUMMARY OF THE INVENTION

The present invention provides compounds of formula (I):

wherein R1-R8 of formula (I) are defined herein below.

The invention also provides methods of selectively activatingchaperone-mediated autophagy (CMA) in a subject in need thereofcomprising administering to the subject a compound of formula (I), or acompound of formula (II), or a combination of a compound of formula (I)and a compound of formula (II), in an amount effective to activate CMA,wherein formula (II) is

wherein R1-R9, X and Y of formula (II) are defined herein below.

The invention also provides methods of protecting cells from oxidativestress, proteotoxicity and/or lipotoxicity in a subject in need thereofcomprising administering to the subject any of the compounds disclosedherein, or a combination of a compound of formula (I) and a compound offormula (II), in an amount effective to protect cells from oxidativestress, proteotoxicity and/or lipotoxicity.

The invention further provides methods of antagonizing activity ofretinoic acid receptor alpha (RARα) in a subject in need thereofcomprising administering to the subject any of the compounds disclosedherein, or a combination of a compound of formula (I) and a compound offormula (II), in an amount effective to act as a RARα antagonist.

The invention also provides methods of screening for a compound thatactivates CMA without affecting macroautophagy, the methods comprisingidentifying a compound that binds to α-helices H12, H3 and H10 ofretinoic acid receptor alpha (RARα), wherein a compound that binds tothe α-helices H12, H3 and H10 of RARα is a candidate compound foractivating CMA without affecting macroautophagy.

The invention further provides methods of screening for a compound thatprotects cells from oxidative stress, proteotoxicity and/orlipotoxicity, the methods comprising identifying a compound that bindsto α-helices H12, H3 and H10 of retinoic acid receptor alpha (RARα),wherein a compound that binds to the α-helices H12. H3 and H10 of RARαis a candidate compound for protecting cells from oxidative stress,proteotoxicity and/or lipotoxicity.

Also provided are pharmaceutical compositions comprising a combinationof a compound of formula (I) and a compound of formula (II) and apharmaceutically acceptable carrier or diluent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-ID. Effect of knock-down of RARα on intracellular turnover oflong-lived proteins. (a) Knock-down of RARα in NIH3T3 mouse fibroblastswas conducted using two different shRNA (c1 and c2). Ctr: control. Left:Representative immunoblot. Actin is shown as loading control. Right:Levels of RARα in control and knock-down cells determined bydensitometric quantification of immunoblots as the one shown on theleft. Values are normalized for actin and expressed as times control(none) values. (n=3) (b) Rates of degradation of long-lived proteins incontrol and RARα knock-down cells maintained in the presence or absenceof serum for 12 h. Values are expressed as percentage of proteolysis.(n=3) (c, d) Percentage of lysosomal (c) and macroautophagy (d)degradation in cells assayed as in B, but treated with inhibitors oflysosomal proteolysis (c) or with 3-methyladenine to blockmacroautophagy (d). Values are expressed as percentage of total proteindegradation sensitive to the lysosomal inhibitors (n=3). All values aremean+S.E. and differences with control are significant for* p<0.05.

FIG. 2A-2C. Effect of knock-down of RARα on macroautophagy. (a)Immunoblot for LC3-II of mouse fibroblasts control (ctr) or knocked-downfor RARα (RARα (−)) maintained in the presence or absence of serum forthe indicated times. Where indicated protease inhibitors (PI) againstlysosomal proteolysis were added. Actin is shown as loading control. (b)Levels of LC3-II determined by densitometric quantification ofimmunoblots. Values are expressed as folds values in serum supplementedcontrol cells (n=4). (c) Ratio of levels of LC3-II in cells treated withPI compared to untreated cells. Values are expressed as fold untreated(n=4). All values are mean+S.E. Differences with control (*) cells aresignificant for *p<0.05.

FIG. 3A-3C. Effect of all-trans-retinoic acid (ATRA) on autophagy. (a)Rates of degradation of long-lived proteins in mouse fibroblastsuntreated (None) or treated with (40 μM) ATRA and maintained in thepresence or absence of serum. Values are expressed as percentage ofproteolysis. (n=3) (b) Percentage of lysosomal degradation calculatedafter treatment with inhibitors of lysosomal proteolysis for 12 h (n=3).(c) Immunoblot for LC3-II of the same cells maintained in the presenceor absence of serum and protease inhibitors (PI). Left: representativeimmunoblot. Actin is shown as loading control. Bottom: Ratio of levelsof LC3-II in cells treated with PI compared to untreated cells. Valuesare expressed as fold untreated (n=4). All values are mean+S.E. anddifferences with untreated cells are significant for * p<0.01.

FIG. 4A-4D. Design, Synthesis and molecular docking of RARα TargetingCompounds. (a) Molecular structure of all-trans-retinoic acid (left)highlighting three different regions: the hydrophobic ring, the polyenelinker “connector”, and the carboxylic acid moiety. The basic structureof the four families of compounds generated through modifications ofATRA using structure-based chemical design strategies are shown.Numbering is shown in the retinoic acid and the α-aminonitrile retinoidbackbone to indicate how these positions have been conserved in the newmolecules. (b) Synthetic reaction schemes for the two novel guanidineretinoids (GR1 and GR2) and the atypical retinoid (AR7). (c-e) Moleculardocking of the AR7 (c), GR1 (d) and GR2 (e) compounds in the RARαbinding pocket. A close view of the RARα binding pocket in ribbon andinteracting residues in stick for each compound docked in thelowest-energy docking pose I is shown. Compounds are docked to ahydrophobic region of the RARα binding pocket formed by α-helices H3,H10 and H12, which is associated with antagonism and blocking of theactive RARα conformation. All compounds form extensive hydrophobicinteractions with hydrophobic residues of the RARα binding pocket.Hydrogen bonds are formed from the guanidinium group of GR1 and GR2 toside-chain hydroxyls of Ser229 and Thr233, and backbone carbonyl oxygenof Pro407.

FIG. 5A-SE. Effect of novel retinoid derivates activators of CMA on RARαactivity. (a-d) Mouse fibroblasts were co-transfected with the hRARαreceptor construct (a, b) or the hRXR receptor (c,d), a relevantreporter luciferase plasmid and the non-retinoid regulated renillareporter to control for transfection. Values show luciferase unitsdetected in cells subjected to: (a, c) the indicated concentrations ofATRA and the three retinoid-derivatives for 12 h. (b, d) I00 nM (b) or10 μM (d) ATRA alone (ATRA) or in the presence of the indicatedconcentrations of the three retinoid derivatives or the antagonistBMS614. Values show luciferase intensity expressed as percentage of thatin cells treated only with ATRA and Ki are shown on the right (n=4-6).(e) Immunoblot for LC3 of cells treated with 20 μM of the retinoidderivatives and protease inhibitors (PI), as labeled. Actin is shown asloading control. Levels of LC3-II in untreated cells (left) and increaseafter PI treatment (LC3-II flux) (right) were calculated from thedensitometric quantification of immunoblots. Values are mean+S.E. (n=3).

FIG. 6A-6C. Characterization of the effect of the novel retinoidderivates on CMA. (a) Rates of degradation of long-lived proteins inmouse fibroblasts control or knocked-down (−) for RARα or for LAMP-2Aand left untreated (None) or treated with (20 μM) the indicatedcompounds. Values are expressed as folds the proteolytic rate inuntreated cells for each group. (n=3) (b-c) Mouse fibroblasts control(Ctr) knocked-down (−) for RARα, LAMP-2A or LAMP-2B were transfectedwith the KFERQ-mcherry1 photoactivable reporter and supplemented or notwith the indicated compounds (20 μM). Average number of fluorescentpuncta per cell quantified in >50 cells in at least 4 different fields.No puncta was detected in LAMP-2A(−) cells. All values are mean+S.E.Differences with untreated samples (*) are significant for * p<0.01.

FIG. 7A-7F. Effect of the novel retinoid derivatives over different CMAcomponents and in the cellular response against different stressors. (a)Rat liver lysosomes treated or not with lysosomal protease inhibitors(PI) were incubated with Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) alone (None) or in the presence of 20 μM of the indicatedcompounds. Immunoblot of samples collected by centrifugation (top) andquantification of the amount of GAPDH bound and taken up by each groupof lysosomes (bottom). (n=3). (b) Immunoblot for the indicated proteinsin homogenates (Hom) and same lysosomes (Lys) as in a. (c) mRNA levelsof LAMP-2A in mouse fibroblasts control (Ctr) or knocked-down (−) forRARα and subjected to the indicated treatments. Values are corrected foractin and are expressed as folds control untreated cells. Differenceswith untreated samples (*) are significant for p<0.01. (n=4-5). (d) Top:Cellular viability of control mouse fibroblasts exposed to 2 mM PQ orLAMP-2A(−) exposed to 0.5 mM PA and treated with the indicated compoundsfor 12 h before or after the PQ treatment. Bottom: Cellular viability ofmouse fibroblasts knocked down for LAMP-2A and treated with 0.5 mM PQ inthe presence of the indicated compounds. (n=3). (e) Viability of mousefibroblasts transfected with the indicated concentrations of a plasmidcoding for α-synuclein and left untreated (none) or treated with 1 mM PQalone or in the presence of 20 μM AR. Differences with untreated cells(*) or with cells treated only with PQ (§) were significant for p<0.001.(n=3). (f) Immunoblot for α-synuclein and actin in cells transfected ornot with α-synuclein, as labeled, and left untreated (none) or treatedwith PQ or PQ and AR7. Top: higher exposure blot to highlight oligomericspecies. * non specific band. M: monomer; Oligo: oligomers). All valuesare mean+S.E.

FIG. 8. Immunoblot for LC3 of cells treated with 20 mM of the retinoidderivatives and protease inhibitors (PI), as labeled. Levels of LC3-1lin untreated cells (left) and increase after PI treatment (LC3-II flux)(right) were calculated from the densitometric quantification ofimmunoblots. Values are mean*S.E. (n=3).

FIG. 9A-9D. Medicinal chemistry refinement for novel atypical retinoidantagonists (ARA). CMA reporter assay in cells treated with AR7derivatives (20 mM for 12 h). (A) High content microscopy images. Nucleiare highlighted with DAPI. (B) Quantification of number of puncta percell relative to AR7 treatment. Compounds with higher CMA activity thanAR7 are marked. (C,D) Dose-dependence (C) and structure (D) of threepreferred compounds.

FIG. 10A-10D. Medicinal chemistry refinement for novel atypical retinoidantagonists (ARA). CMA reporter assay in cells treated with AR7derivatives (20 mM for 12 h). (A) High content microscopy images. Nucleiare highlighted with DAPI. (B) Quantification of number of puncta percell relative to AR7 treatment. (C) Structure of the original molecule(AR7) and two preferred compounds regarding potency and selectivity. (D)Dose-dependence of the original molecule and two preferred compounds.Values are mean+s.e.m. n=3 experiments with triplicate replica.

FIG. 11. CMA activation in different cell types by atypical retinoidantagonists (ARA). CMA reporter assay in the indicated cell linestreated with AR7 and its derivative QX39 (20 mM for 12 h). Values aremean+s.e.m. n=3 experiments with triplicate replica.

FIG. 12A-12C. Protective effect of atypical retinoid antagonists (ARA).(A) Experimental design: NIH3T3 cells were exposed to increasingconcentrations of paraquat (PQ) to induce oxidative stress. ARA whereadded in conjunction with the stressor or where indicated, also beforethe stressor. (B-C) Analysis of cell viability in the two experimentalparadigms shown in (A), to show that whereas the original ARA need to beadded before the insult to have maximal protective effect, the new ARAshow protection even without pre-treatment. Values are mean+s.e.m. n=3independent experiments with triplicate wells. *p<0.01

FIG. 13A-13B. Protective effect of atypical retinoid antagonists (ARA)against different stressors in different cell types. Mouse embryonicfibroblasts (A) or neuroblastoma cell lines (B) were subjected to theindicated stressors in the presence or not of the two ARA. Viability wasmeasure at 24 h of the treatment and the protective effect wascalculated as the increase in cell viability compared to untreatedcells. Values are mean+s.e.m. of n=3 independent experiments withtriplicate samples. All values were significantly different (p<0.01) tovalues in untreated samples.

FIG. 14. Medicinal chemistry refinement for atypical retinoidantagonists (ARA). CMA reporter assay in cells maintained in thepresence (top) or absence (bottom) of serum and treated with AR7derivatives (20 mM for 12 h). Quantification of number of puncta percell relative to AR7.

FIG. 15A-15C. Medicinal chemistry refinement for atypical retinoidantagonists (ARA). (A) Structure of ARA. (B-C) CMA reporter assay incells treated with AR7 derivatives (20 mM for 12 h) while maintained inthe presence (B) or absence (C) of serum. Values are mean+s.e.m. n=3experiments with triplicate replica.

FIG. 16A-16B. Effect of atypical retinoid antagonists (ARA) on totalproteolysis and lysosomal degradation measured at difference time points(6 h, 12, and 24 h) in the presence or absence of serum. Values aremean+s.e.m. of n=3 independent experiments with triplicate samples.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a compound having the structure of formula (I)

whereinR1 and R2 of formula (I) are independently H or methyl:R4, R5, R6 and R7 of formula (I) are independently, H, hydroxyl, halogenor alkyl, or R8 and R5 or R6 of formula (I) together form a 5- or6-membered heteroaryl; andR8 of formula (I) is C≡N, 5- or 6-membered heteroaryl.

where “a” represents the point of attachment of R8 to the 6-memberedring, and Q is a 5- or 6-membered heteroaryl or Q and C═O of R8 togetherform a 5- or 6-membered heteroaryl, or R8 and R5 or R6 of formula (I)together form a 5- or 6-membered heteroaryl, where each heteroaryl canbe optionally substituted with one or more of CN, ═O, NH₂ and phenyl: ora pharmaceutically acceptable salt thereof. Preferably, the halogen isBr, Cl, F or I. Preferably, the alkyl is C1-C3 alkyl.

Pharmaceutically acceptable salts that can be used with compounds of thepresent invention are non-toxic salts derived, for example, frominorganic or organic acids including, but not limited to, salts derivedfrom hydrochloric, sulfuric, phosphoric, acetic, lactic, fumaric,succinic, tartaric, gluconic, citric, methanesulphonic andp-toluenesulphonic acids.

Preferred compounds include those having a structure selected from thegroup consisting of:

or a combination thereof, or a pharmaceutically acceptable salt thereof.

Also provided is a pharmaceutical composition comprising any of thecompounds disclosed herein, or a combination of any compounds disclosedherein, and a pharmaceutically acceptable carrier or diluent. Examplesinclude a combination of a compound of formula (I) and a compound offormula (II) and a pharmaceutically acceptable carrier or diluent,wherein formula (II) is

wherein R1, R2, R3, R4, R5, R6, R8 and R9 of formula (II) areindependently H, hydroxyl, halogen, SH, NO₂, CF₃, COOH, COOR10, CHO, CN,NH₂, NHR10, NHCONH₂, NHCONHR10, NHCOR10, NHSO₂R10, OCR10, COR10, CH₂R10,CON(R10,R11), CH═N—OR10, CH═NR10, OR10, SR10, SOR10, SO₂R10, COOR10,CH₂N(R10,R11), N(R10,R11), or optionally substituted lower alkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalky, aryl, heteroaryl,aralkyl, or heteroaralkyl; wherein the optional substituent is one ormore of F, Cl, Br, I, OH, SH, NO₂, COOH, COOR10, R10, CHO, CN, NH₂,NHR10, NHCONH₂, NHCONHR10, NHCOR10, NHSO₂R10, HOCR10, COR10, CH₂R10,CON(R10, R11), CH═N—OR10, CH═NR10, OR10, SR10, SOR10, SO₂R10, COOR10,CH₂N(R10, R11), N(R10, R11);wherein R7 of formula (II) is H, hydroxyl, halogen, CF₃, CN, OCF₃, COOH,COOCH₃, COOR10, COO(CH₂)₂Si(CH₃)₃, COOR10Si(CH₃)₃, NHCOCH₃, C≡C—CH₂OH,C≡C—R10-OH or optionally substituted alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cyclic or heterocyclic;wherein the optional substituent is one or more of F, Cl, Br, I, OH, SH,NO₂, CH₃, R10, COOH, COOR10, CHO, CN, NH₂, NHR10, NHCONH₂, NHCONHR10,NHCOR10, NHSO₂R10, HOCR10, COR10, CH₂R10, CON(R10, R11), CH═N—OR10,CH═NR10, OR10, SR10, SOR10, SO₂R10, COOR10, CH₂N(R10, R11), N(R10, R11);wherein R10 and R11 are independently H or C1-C6 alkyl;wherein X is C, C═O, N, O, S or S═O; and Y is N, NH or C: or apharmaceutically acceptable salt thereof.

Synthesis of compounds of formula (II) is described below and in U.S.Patent Application Publication No. 2011/0251189, published Oct. 13,2011, the contents of which is herein incorporated by reference in itsentirety.

Pharmaceutically acceptable carriers and diluents that can be usedherewith encompasses any of the standard pharmaceutical carriers ordiluents, such as, for example, a sterile isotonic saline, phosphatebuffered saline solution, water, and emulsions, such as an oil/water orwater/oil emulsions.

The invention also provides a method of selectively activatingchaperone-mediated autophagy (CMA) in a subject in need thereofcomprising administering to the subject any of the compounds disclosedherein, or a compound of formula (II), or a combination of a compound offormula (I) and a compound of formula (II), in an amount effective toactivate CMA, wherein formula (II) is

wherein R1, R2, R3, R4, R5, R6, R8 and R9 of formula (II) areindependently H, hydroxyl, halogen, SH, NO₂, CF₃, COOH, COOR10, CHO, CN.NH₂, NHR10, NHCONH₂, NHCONHR10, NHCOR10, NHSO₂R10, OCR10, COR10, CH₂R10,CON(R10,R11), CH═N—OR10, CH═NR10, OR10, SR10, SOR10, SO₂R10, COOR10,CH₂N(R10,R11), N(R10,R11), or optionally substituted lower alkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalky, aryl, heteroaryl,aralkyl, or heteroaralkyl; wherein the optional substituent is one ormore of F, Cl, Br, I, OH, SH, NO₂, COOH, COOR10, R10, CHO, CN, NH₂,NHR10, NHCONH₂, NHCONHR10, NHCOR10, NHSO₂R10, HOCR10, COR10, CH₂R10,CON(R10, R11), CH═N—OR10, CH═NR10, OR10, SR10, SOR10, SO₂R10, COOR10,CH₂N(R10, R11), N(R10, R11);wherein R7 of formula (II) is H, hydroxyl, halogen, CF₃, CN, OCF₃, COOH,COOCH₃, COOR10, COO(CH₂)₂Si(CH₃)₃, COOR10Si(CH₃), NHCOCH₃, C≡C—CH₂OH,C≡C—R10-OH or optionally substituted alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cyclic or heterocyclic;wherein the optional substituent is one or more of F, Cl, Br, I, OH, SH,NO₂, CH, R10, COOH, COOR10, CHO, CN, NH₂, NHR10, NHCONH₂, NHCONHR10,NHCOR10, NHSO₂R10, HOCR10, COR10, CH₂R10, CON(R10, R11), CH═N—OR10,CH═NR10, OR10, SR10, SOR10, SO₂R10, COOR10, CH₂N(R10, R11), N(R10, R11):wherein R10 and R11 are independently H or C1-C6 alkyl;and wherein X is C, C═O, N, O, S or S═O: and Y is N, NH or C; or apharmaceutically acceptable salt thereof. The subject can have, forexample, a neurological disease or disorder, a neurodegenerativedisease, a tauopathy, Parkinson's Disease, Alzheimer's Disease,Huntington's Disease, frontotemporal dementia, retinal degeneration,multiple sclerosis, diabetes, a lysosomal storage disorder, a retinaldisease, a cardiovascular disease, Myocardial infarction, cardiachypertrophy or a cardiomyopathy. The subject can have reduced CMAcompared to a normal subject prior to administering the compound.Preferably, the compound does not affect macroautophagy or otherautophagic pathways. In macrophagy, proteins and organelles aresequestered in double-membrane vesicles and delivered to lysosomes fordegradation. In CMA, protein substrates are selectively identified andtargeted to the lysosome via interactions with a cytosolic chaperone.

The invention also provides a method of protecting cells from oxidativestress, proteotoxicity and/or lipotoxicity in a subject in need thereofcomprising administering to the subject any of the compounds disclosedherein, or a combination of a compound of formula (I) and a compound offormula (II), in an amount effective to protect cells from oxidativestress, proteotoxicity and/or lipotoxicity. The subject can have, forexample, one or more of the chronic conditions that have been associatedwith increased oxidative stress and oxidation and a background ofpropensity to proteotoxicity. The subject can have, for example, one ormore of a neurological disease or disorder, a neurodegenerative disease,a tauopathy, Parkinson's Disease, Alzheimers Disease, Huntington'sDisease, frontotemporal dementia, retinal degeneration, multiplesclerosis, diabetes, a lysosomal storage disorder, a cardiovasculardisease, myocardial infarction, cardiac hypertrophy and acardiomyopathy. The cells being protected can comprise, for example,cardiac cells, liver cells, neurons, myocytes, fibroblasts and/or immunecells. The compound can, for example, selectively activatechaperone-mediated autophagy (CMA). In one embodiment, the compound doesnot affect macroautophagy. The compound can, for example, antagonizeactivity of retinoic acid receptor alpha (RARα).

The invention also provides a method of antagonizing activity ofretinoic acid receptor alpha (RARα) in a subject in need thereofcomprising administering to the subject any of the compounds disclosedherein, or a combination of a compound of formula (I) and a compound offormula (II), in an amount effective to act as a RARα antagonist.

Preferred embodiments include those where the compound of formula (II)has the formula

or a pharmaceutically acceptable salt thereof.

Also provided is a compound having the structure

whereinR1, R2, R3, R4, R5, R6, R8 and R9 are independently H, hydroxyl,halogen, CF₃, COOH, or COOCH₃, SH, NO₂, COOR10, CHO, CN, NH₂, NHR10,NHCONH₂, NHCONHR10, NHCOR10, NHSO₂R10, OCR10, COR10, CH₂R10,CON(R10,R11), CH═N—OR10, CH═NR10, OR10, SR10, SOR10, SO₂R10, COOR10,CH₂N(R10,R11), N(R10,R11), or optionally substituted lower alkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalky, aryl, heteroaryl,aralkyl, or heteroaralkyl: wherein the optional substituent is one ormore of F, Cl, Br, I, OH, SH, NO₂, COOH, COOR10, R10, CHO, CN, NH₂,NHR10, NHCONH₂, NHCONHR10, NHCOR10, NHSO₂R10. HOCR10, COR10, CH₂R10,CON(R10, R11), CH—N—OR10, CH═NR10, OR10, SR10, SOR10, SO₂R10, COOR10,CH₂N(R10, R11), N(R10, R11);R7 is CF₃, CN, OCF₃, COOH, COOCH₃, COOR10, CO(CH₂)₂Si(CH₃),COOR10Si(CH₃)₃, NHCOCH₃, C≡C—CH₂OH, C≡C—R10-OH or optionally substitutedaryl, heteroaryl, aralkyl, heteroaralkyl, cyclic or heterocyclic:wherein the optional substituent is one or more of F, Cl, Br, I, OH, SH,NO₂, CH₃, R10, COOH, COOR10, CHO, CN, NH₂, NHR10, NHCONH₂, NHCONHR10,NHCOR10, NHSO₂R10, HOCR10, COR10, CH₂R10, CON(R10, R11), CH═N—OR10,CH═NR10, OR10, SR10, SOR10, SO₂R10, COOR10, CH₂N(R10, R11), N(R10, R11);andR10 and R11 are independently H or C1-C6 alkyl; or a pharmaceuticallyacceptable salt thereof.

In any of the methods or compounds or compositions disclosed herein, anyone or more halogen can be Br, Cl, F or I independently of any otherhalogen. In any of the methods or compounds disclosed herein, any one ormore alkyl can be, e.g., C1-C6 alkyl or C1-C3 alkyl independently of anyother alkyl. In any of the methods or compounds disclosed herein, anyone or more aralkyl can contain C1-C3 alkyl independently of any otheraralkyl. An alkyl can be, for example, methyl, ethyl, propyl, butyl,pentyl, or hexyl, independently of any other alkyl.

In any of the methods or compounds or compositions disclosed herein, theoptionally substituted aryl or heteroaryl can be

where the wavy line

indicates the point of attachment of the optionally substituted aryl orheteroaryl to the main structure.

Preferred compounds include the following compounds:

or a combination thereof, or a pharmaceutically acceptable salt thereof.

The invention also provides a method of screening for a compound thatactivates CMA without affecting macroautophagy, the method comprisingidentifying a compound that binds to α-helices H12, H3 and H10 ofretinoic acid receptor alpha (RARα), wherein a compound that binds tothe α-helices H12, H3 and H10 of RARα is a candidate compound foractivating CMA without affecting macroautophagy. A photoconvertiblefluorescent reporter assay to track CMA has been described.³⁰

The invention further provides a method of screening for a compound thatprotects cells from oxidative stress, proteotoxicity and/orlipotoxicity, the method comprising identifying a compound that binds toα-helices H12, H3 and H10 of retinoic acid receptor alpha (RARα).Wherein a compound that binds to the α-helices H12, H3 and H10 of RARαis a candidate compound for protecting cells from oxidative stress,proteotoxicity and/or lipotoxicity. The compound can protect cells fromoxidative stress, proteotoxicity and/or lipotoxicity through activationof CMA.

H3. H10 and H12 of human RARα have the following amino acid sequences:H3: DIDLWDKFSELSTKCIIKTVEFAK (SEQ ID NO:1), H10: DLRSISAKGAERVITLKMEIP(SEQ ID NO:2) and H12: GSMPPLIQEMLEN (SEQ ID NO:3).

In either of the above two screening methods, the compound can be, e.g.,a retinoic acid receptor α (RARα) antagonist.

The compounds and compositions of the present invention can beadministered to subjects using routes of administration known in theart. The administration can be systemic or localized to a specific site.Routes of administration include, but are not limited to, intravenous,intramuscular, intrathecal or subcutaneous injection, oral or rectaladministration, and injection into a specific site.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS Example A Overview

The effect of retinoic acid receptor (RAR) signaling on CMA activity wasinvestigated by taking advantage of expertise in the design andsynthesis of novel retinoid derivatives with different biologicalactivities^(23,24) to generate novel RARα modulators that could be usedto modify CMA activity. Structure-based chemical design was used tomimic the critical regions of all-trans-retinoic acid (ATRA), andmodifications were introduced that allow these novel retinoids toselectively modulate different RARα downstream effects. These compoundswere demonstrated to lead to selective activation of CMA withoutnoticeable changes in other autophagic pathways. Furthermore, it wasdemonstrated that chemical enhancement of CMA by treatment of cells withthese novel compounds renders cells more resistant to oxidative stressand proteotoxicity. These findings highlight the therapeuticapplicability of these and related compounds in the treatment of chronicdiseases that associate with loss of CMA activity.

Methods

Animals, cells and reagents. Adult male Wistar rats (Charles RiverLaboratories) fasted for 48 h before sacrifice were used for isolationof lysosomes from liver. All animal work was approved and performedaccording to the guidelines set by the Albert Einstein College ofMedicine Institutional Animal Care and Use Committee. Mouse fibroblasts(NIH3T3) from the American Type Culture Collection were cultured inDulbecco's Modified Eagle's Medium (Sigma) in the presence of 10%newborn calf serum. Serum removal was performed by thoroughly washingthe cells with Hanks' Balanced Salt Solution (Invitrogen) and placingthem in serum-free complete medium. Where indicated, cells were treatedwith the macroautophagy inhibitor 3-methyladenine (Sigma) at a finalconcentration of 10 mM or with 20 mM NH₄Cl and 100 μM leupeptin (FisherBioReagents) to inhibit lysosomal proteolysis. Where indicated, paraquatwas added directly to the culture media to induce oxidative stress.Stable knock-down of LAMP-2A, LAMP-2B or RARα was obtained usingvector-mediated stable RNA interference (RNAi) directed specificallyagainst the LAMP-2A or LAMP-2B exon as described previously³³ or againstthe two following regions of human RARα: GAAAGTCTACGTCCGGAAA (SEQ IDNO:4) and GCAGCAGTTCCGAAGAGAT (SEQ ID NO:5). The plasmid encoding formCherry-GFP-LC3 was from Addgene and for α-synuclein was a generous giftfrom Dr. Esther Wong (Navang Technological University, Singapore).Sources of chemicals and antibodies were as describedpreviously^(8,9,30,35). ATRA was purchased from Sigma, BMS614 and AM580were from Tocris Bioscience and the antibody against RARα was from CellSignaling.

Autophagic measurements. Intracellular protein degradation was measuredby metabolic labeling and pulse chase experiments as described before³⁹.Autophagic flux was measured as changes in levels of LC3-II uponinhibition of lysosomal proteolysis²⁹ and as the ratio mcherry-positivepuncta to double labelled (mCherry-GFP) puncta in cells transfected withthe mCherry-GFP-LC3 reporter⁴⁰. CMA activity was determined using thephotoactivable KFERQ-PA-mCherry reporter that allows visualization ofCMA activation as an increase in the number of fluorescent puncta percell³⁰. Analysis of CMA in isolated lysosomes was performed using apreviously developed in vitro assay to measure the ability of intactlysosomes to take up and degrade well-characterized CMA substrateproteins³⁹. Lysosomes active for CMA were isolated from rat liver usingdifferential centrifugation and floatation in discontinuous densitymetrizamide gradients following a previously optimized procedure⁴¹.Lysosomes from cultured cells were isolated by similar procedures butusing instead discontinuous metrizamide/percoll gradients after ruptureof the plasma membrane through nitrogen cavitation³³.

Design and synthesis of novel-retinoid derivatives. Preparation ofguanidine retinoids (GR1) and (GR2) (FIG. 4b and Table 1): Solid sodiumchloride obtained from reacting guanidine hydrochloride in DMF:dioxaneand sodium tert-butoxide under nitrogen (g) at 50-55° C. for 30 min wasfiltered and the filtrate was added to a solution of retinoid and CDI inDMF. The progress of the reaction was monitored by TLC, the solidproduct was collected by filtration and washed to remove excessguanidine.

Preparation of atypical retinoid AR7 (FIG. 4b and Table 1): The2H-benzo[b][1,4] oxazines were synthesized by modifying the existingmethods⁴². 2-bromo-4-chloroacetophenone (0.01 mol) in dichloromethanewas added drop-wise to a solution of 2-aminophenol in dichloromethane,aqueous potassium carbonate (20% w/v) and tetrabutylammonium hydrogensulphate. The resultant mixture was refluxed till completion for 4-6 hand the organic layer was extracted with dichloromethane and dried oversodium sulphate evaporated in vacuum to give a crude solid product. Thesolid was then recrystallized with hot ethanol to obtain pure yield87-95%. In the NOE analysis, E/Z isomers of GR1 were assigned based on apair of weak vinyl peaks at 6.4 ppm and 6.12 ppm (Z-isomer) and anotherpair of strong vinyl peaks at 6.27 ppm and 5.98 ppm (E-isomer). For GR2,E/Z isomers were assigned based on a pair of weak vinyl peaks at 6.43ppm and 6.12 ppm (Z-isomer) and another pair of strong vinyl peaks at6.19 ppm and 6.00 ppm (E-isomer).

In silico docking. AR7, GR1 and GR2 structures were drawn in ChemDrawUltra 12.0 and converted to three-dimensional all-atom structures fromsdf format using LigPrep (Version 2.5, Schrödinger, LLC, New York, N.Y.,2011). For each ligand a maximum of 4 stereoisomers were generated,ionization states and tautomers were generated for pH 7 and pH 2 andgeometries optimized and energy minimized before for docking. Thestructure of the RARα-RXR hetero-dimer in complex with the smallmolecule antagonist BM614 (PDB ID: 1DKF), was used for docking andmolecular dynamics. The RARα-RXR structure was prepared using MAESTROprotein preparations module (Version 9.2, Schrödinger, LLC, New York,N.Y. 2011). The structure of the antagonist was removed from the RARαsite, water molecules at a distance of more than 5 Å from heteroatomswere removed, all missing protons were generated, hydrogens wereoptimized for best hydrogen bonding network bonds and formal chargeswere assigned and structure was gently minimized by restrained energyminimization. The ligand-binding pocket was defined within 5 Å of theBMS614 pose and receptor grid size and center was generated based on theposition and the size of the BMS614. To account for receptor flexibilityin docking, scaling of van der Waals' radii of non polar atoms with theabsolute value of the partial atomic charge less than or equal to 0.25for protein atoms was set to 1 and for ligand non polar atoms withpartial atomic charges less than or equal to 0.15 was set to 0.8.Docking was performed in ligand flexible mode using Glide⁴³⁻⁴⁵ (Version5.8, Schrödinger) using the extra precision (XP) mode. All threemolecules were docked into the BMS614 binding site with and withoutrotatable binding site hydroxyl-groups. Structures were analyzed usingMAESTRO and PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4Schrödinger, LLC.)

Molecular Dynamics Simulations. The lowest-energy docked structures foreach ligand were minimized using the Desmond molecular dynamicssystem⁴⁶⁻⁴⁸ (Version 3.0, D. E. Shaw Research, New York, N.Y., 2011.Maestro-Desmond Interoperability Tools, version 3.1, Schrödinger. NewYork, N.Y., 2012). All minimization and simulation systems were set upusing MAESTRO tools. The minimization protocol was performed using anOPLS-2005 force field with a cubic-TIP3P water-box, 150 mM NaCl andwater-box boundaries set to a minimum of 10 A from the proteins surfacein all directions. Minimization was performed with a maximum of 2000iterations with a convergence threshold of 1 kcal/mol/Å, a steepestdescent method was used initially until a gradient threshold of 25kcal/mol/Å is reached. Simulated annealing and molecular dynamics inexplicit water were performed on the refined lowest energy dockedstructures, using the Desmond molecular dynamics system and OPLS-2005force field. A five stage simulated annealing protocol was performedwith 40 ps intervals where the temperature was linearly interpolatedbetween each time point with temperature steps of 10, 100, 300, 400, and300 K. An NVT ensemble class was employed with a Berendsen thermostatwith a relaxation time of 1 ps. A 6 ns molecular dynamics simulation wasperformed with and without simulated annealing for each of the minimizedlowest-energy docking posses. Molecular dynamics simulations wereperformed using an NPT ensemble class at 300 K and 1.01325 bar. ANose-Hoover chain thermostat method with a 1 ps relaxation time and aMartyna-Tobias-Klein Barostat Method with a 2 ps relaxation time wereused. All docking and molecular dynamic simulations were analyzed usingtools within MAESTRO and figures were prepared using PyMOL.

Measurement of RARα and RXR activity. A RAR-responsive luciferaseconstruct was utilized to monitor the activity of RARα in culturedcells. Cells were co-transfected with pCMX-Gal-L-hRARα and a constructencoding the firefly luciferase reporter gene under the control of aminimal promoter and tandem repeats of the retinoic-acid responseelement (tk-px3-luc). Co-transfection with a renilla luciferase reporterplasmid was performed to control for efficiency of transfection. RXRactivity was measured by similar procedures using co-transfection withpCMX-mRXR and tk-apoA1-luc. Compounds were added 24 h after transfectionand 48 h later cells were lysed and assayed for luciferase activityusing the Dual-Luciferase Reporter Assay System (Progmega). Luciferasevalues were normalized to the renilla luciferase reporter.

Intracellular protein degradation. Confluent cells labelled with[³H]leucine (2□Ci/ml) (NEN-PerkinElmer Life Sciences) for 48 h wereextensively washed and maintained in medium with an excess of unlabeledleucine⁴⁹. Aliquots of the medium taken at different times wereprecipitated in trichloroacetic acid and proteolysis measured as theamount of acid-precipitable radioactivity transformed in acid-solubleradioactivity at each time. The amount of lysosomal proteolysis wascalculated by treating parallel wells with 20 mM NH₄Cl and 100 μMleupeptin during the chase period.

CMA reporter assay. The photoactivable CMA reporter was constructed byinserting a sequence of 21 amino acids of Ribonuclease A bearing theCMA-targeting motif in the N-terminus multicloning site of thephotoactivable protein mCherry1 (PA-mCherry1)³⁰. Cells transduced with alentivirus carrying the KFERQ-bearing constructs were photoactivated byexposure to a 3.5 mA (current constant) light emitting diode (LED:Norlux, 405 nm) for 10 min and at the desired times fixed in 3%formaldehyde and images were captured with an Axiovert 200 fluorescencemicroscope (Zeiss) with apotome and equipped with a 63× 1.4 NA oilobjective lens and red (ex. 570/30 nm, em. 615/30 nm), cyan (ex. 365/50nm and em. 530/45 nm) and green (ex. 475/40 nm and em. 535/45 nm) filersets (Chroma). All images were acquired with a high-resolution CCDcamera after optical sectioning through the apotome. Images wereprepared using Adobe Photoshop 6.0 software (Adobe Systems).Quantification was performed in individual frames after deconvolutionand thresholding using ImageJ software (NIH) in a minimum of 50 cells.

Lysosomal in vitro uptake assay. Transport of purified proteins intoisolated lysosomes was analyzed using a previously described in vitrosystem⁴¹. Briefly, intact lysosomes treated or not with a pool ofprotease inhibitors for 10 min on ice, were incubated withglyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 20 min at 37° C. Atthe end of the incubation, lysosomes were recovered by centrifugation,washed, and subjected to SDS-PAGE and immunoblot for GAPDH. Binding wascalculated after densitometric analysis as the amount of GAPDH recoveredin the lysosomes not treated with protease inhibitors, and uptake as thedifference in the amount of GAPDH recovered in treated minus untreatedlysosomes.

Lentivirus-mediated shRNA. Lentiviral particles were generated byco-transfection with the lentiviral transfer vector carrying the hairpinsequence against the desired mRNA and the third-generation packagingconstructs pMDLg/pRRE and pRSV-REV, and as envelope the G glycoproteinof the VSV (pMD2.G) into HEK293T cells as described before³³. Culturedcells were transduced by addition of packed virus at a titter of2.63×10⁶ units/ml.

General procedure for preparation of guanidine retinoids (GR1 and GR2)²³(FIG. 4b and Table 1): A solution of guanidine hydrochloride (2 mmol) inDMF:dioxane (1:1:5 ml) was added sodium tert-butoxide (2 mmol) and thereaction mixture was heated under nitrogen at 50-55° C. for 30 min. Themixture was cooled to room temperature, the solid sodium chloride wasfiltered and the filtrate was added to the 1 h stirred solution ofretinoid and CDI in DMF at room temperature. The progress of thereaction was monitored by TLC. After completion of the reaction, water(10 ml) was added, the solid product was collected by filtration and thesolid was washed with cold water to remove excess guanidine.

Compound GR1:N-[4-(3,5,5-trimethyl-cyclohex-2-enylidenemethyl)-benzoyl]-guanidine. 1HNMR (300 MHz, CDCl3): δ 0.88 (s, 6H), 1.78 (s, 3H), 1.91 (s, 2H), 2.33(s, 2H), 2.48-2.53 (m, 4H), 2.74 (s, 1H), 2.90 (s, 1H), 3.52 (s, 1H),6.01 (s, 1H), 6.32 (s, 1H), 7.24-7.28 (d, J=12 Hz, 2H), 8.01-8.05 (d,J=12 Hz, 2H); 13C NMR (75 MHz, CDCl3): δ 25.0, 29.1, 31.2, 45.1, 126.0,127.3, 128.8, 129.5, 137.6, 137.9, 138.0, 140.6, 163.2, 176.5; HR-MS:(C18H24N3O) calcd ([M+H]+) 298.1921; found 298.1928.

Compound GR2:N-[4-(3-methyl-cyclohex-2-enylidenemethyl)-benzoyl]-guanidine. 1H NMR(300 MHz, CDCl3): δ 1.57-1.1.70 (m, 2H), 1.80 (s, 3H), 2.03-2.13 (m,2H), 2.48-2.60 (m, 6H), 6.01 (s, 1H), 6.20 (s, 1H), 7.23-7.27 (d, J=12Hz, 2H), 7.98-7.02 (d, J=12 Hz, 2H); 13C NMR (75 MHz, CDCl3): δ 23.2,24.7, 26.8, 30.7, 124.5, 127.9, 128.8, 129.3, 137.4, 139.3, 140.2,140.7, 142.7, 163.8, 176.4; HR-MS: (C₁₇H₂₀N₃O) calcd ([M+H]+) 270.1606;found 270.1614.

General procedure for the preparation of atypical retinoids (AR1-AR10)⁵⁰(FIG. 4b and Table 1): The 2H-benzo[b][1,4] oxazines were synthesized bymodifying the existing methods. To a solution of 2-aminophenol (0.001mol) in dichloromethane (40 ml) aqueous potassium carbonate (20% w/v)and tetrabutylammonium hydrogen sulfate (0.0005 mol) was added andmixture was stirred for 2 h at room temperature After 2 h,2-bromo-4-chloroacetophenone (0.01 mol) in 20 ml dichloromethane wasadded drop-wise through a course of 15 min and the resultant mixture wasrefluxed till completion for 4-6 h. The organic layer was extracted withdichloromethane and dried over sodium sulfate evaporated in vacuum togive crude solid product. The solid was then recrystallized with hotethanol to get pure yield 87-95%.

General procedure for the preparation of α-aminonitrile functionalizednovel constrained retinoids (αAmR1-αAmR11)³² (Table 1): Into a 10-mLround-bottomed flask were added β-cyclocitral (1.0 mmol), amine (1.0mmol), TMSCN (1.2 mmol), H₂O (2 mL), and InCl₃ (0.1 mmol) sequentially.The reaction mixture was stirred vigorously at room temperature and theprogress of the reaction was monitored by TLC. After stirring for 4-6 hat room temperature the solid that was obtained was filtered and washedwith water and hexane to yield the desired product retinoids(αAmR1-αAmR11).

General procedure for the preparation of boron-α-aminonitrilefunctionalized novel constrained retinoids (BAmR1-BamR6) (Table 1): Intoa 10-mL round-bottomed flask were added aldehyde (1.0 mmol), amine (1.0mmol), TMSCN (1.2 mmol), H₂O (2 mL), and InCl₃ (0.1 mmol) sequentially.The reaction mixture was stirred vigorously at room temperature and theprogress of the reaction was monitored by TLC. After stirring for 4-6 hat room temperature the solid that was obtained was filtered and washedwith water and hexane to yield the desired product retinoids(BAmR1-BamR6).

General methods. Protein concentration was measured by the Lowry methodusing bovine serum albumin as a standard. Cell viability was determinedusing the CellTiter-Blue® Kit (Promega). Carbonyl groups were detectedwith the OxyBlot Oxidized Protein Detection Kit (ChemiconInternational). Apoptosis was determined with Annexin V-PE apoptosisdetection kit (BD Pharmingen). After SDS-PAGE and immunoblotting, theproteins recognized by the specific antibodies were visualized bychemiluminescence methods (Western Lightning: PerkinElmer) usingperoxidase-conjugated secondary antibodies. Densitometric quantificationof the immunoblotted membranes was performed using Image J (NIH).Quantitative real time PCR was used to determine changes in mRNA levelsusing the TaqMan One-Step RT-PCR Master Mix reagent (Applied Biosystem).Fluorescence was performed using conventional procedures and all imageswere captured with an Axiovert 200 fluorescence microscope (Zeiss) withapotome.

Statistical analysis. All numerical results are reported as mean±s.e.m,and represent data from a minimum of three independent experimentsunless otherwise stated. Statistical significance of difference betweengroups was determined in instances of single comparisons by thetwo-tailed unpaired Student's t-test of the means. In instances ofmultiple means comparisons, we used one-way analysis of variance (ANOVA)followed by the Bonferroni post hoc test to determine statisticalsignificance. Statistic analysis was performed in all of the assays, andsignificant differences are noted in the graphical representations.

Results

RAR signaling has opposite effects in different autophagic pathways.Recent studies have described that retinoic acid exerts a cell-typedependent stimulatory effect on macroautophagy via Beclin-1 upregulationand inhibition of the mTOR pathway²⁵⁻²⁷ or by enhancing autophagosomematuration²⁸. The effects of these interventions on CMA have not beenexplored until now. To directly analyze the effect of RAR signaling ondifferent autophagic pathways. RARα was knocked down in mousefibroblasts, the most abundant type of RAR in these cells. Lentiviraltransduction with two different shRNA against RARα resulted in 75-90%stable knock-down of this receptor (FIG. 1a ).

Analysis of the rates of degradation of long-lived proteins, typicalautophagy substrates, in control and knock-down cells revealed anincrease in protein degradation in RARα (−) cells, when compared tocontrol mouse fibroblasts (FIG. 1b ). This increase was evident bothunder basal conditions and when serum was removed from the culture mediato upregulate autophagy. Addition of lysosomal inhibitors demonstratedthat most of the increase in both basal and inducible proteindegradation in RARα (−) cells was of lysosomal origin (FIG. 1c ).However, the percentage of protein degradation sensitive to3-methyladenine, a well-characterized inhibitor of macroautophagy, wassignificantly reduced in RARα (−) cells (FIG. 1d ), suggesting that theobserved increase in lysosomal degradation was not attributable toupregulation of macroautophagy.

To directly analyze macroautophagy, a widely accepted assay was used tomeasure activity of this pathway based on the analysis of theintracellular levels and degradation of the lipid-conjugated form of thelight chain protein type 3 (LC3-II), a constitutive component ofautophagosomes²⁹. Steady-state levels of LC3-II provide information onthe amount of autophagosomes present in a cell at a given time, whereasthe amount of LC3-II that accumulates upon blockage of lysosomalproteolysis allows measuring the efficiency of fusion and degradation ofautophagic vacuoles by lysosomes (autophagic flux). RARα (−) cellsdisplayed significantly higher levels of LC3-II both under basal andinducible conditions when compared with control cells (FIGS. 2a and b ).Immunofluorescence for LC3 in these cells confirmed the presence ofhigher content of LC3-positive vesicles in the cells defective for RARα.These vesicles were confirmed to be autophagosomes using a double taggedform of LC3 (mCherry-GPF-LC3) that due to quenching of GFP fluorescenceat low pH highlights autophagosomes in yellow and lysosomes in red. Thecontent of double labeled vesicles was significantly higher in RARα (−)cells when compared with control cells. An increase in autophagosomecontent can result from increased formation of these organelles(autophagosome induction) or from reduced clearance of autophagosomes bylysosomes. To differentiate between both possibilities, the autophagicflux was compared in control and RARα (−) cells. Immunoblot (FIGS. 2aand c ) and immunofluorescence for endogenous LC3 revealed significantlyreduced increase in levels of the autophagosome-associated form of thisprotein upon blockage of lysosomal degradation and a reduction in thenumber of single labeled vesicles (lysosomes) when using the doubletagged form of LC3. These results support that elimination of signalingthrough the RARα receptor reduces macroautophagy activity, in agreementwith the previously described stimulatory effect of retinoic acid onthis autophagic pathways.^(25,27,28).

Because the observed downregulation of macroautophagy upon RARαknock-down cannot explain the increase in lysosomal degradation observedin these cells (FIG. 1c ), the effect of this intervention on CMA wasmeasured. Activation of this selective autophagic pathway can bedetermined using a photoactivable (PA) reporter fused to theCMA-targeting motif (KFERQ-PA-mcherry1)³⁰. Activation of CMA favorsmobilization of this artificial CMA substrate from the cytosol tolysosomes, which can be tracked as a change in the reporter fluorescencefrom a diffuse to a punctate pattern³⁰. Fluorescence analyses of cellstransfected with the CMA reporter revealed a significantly higher numberof fluorescent puncta per cell in RARα (−) cells, both in the presenceor absence of serum when compared to control cells. These resultssuggest that the increase in lysosomal degradation observed upon RARαblockage was, for the most part, a consequence of CMA upregulation, andsupport an inhibitory effect of RARα signaling on both basal andinducible CMA.

To further confirm the opposite effect of RARα signaling on differentautophagic pathways and the possible inhibitory effect of signalingthrough this receptor on CMA, similar experiments were performed incells treated or not with all-trans-retinoic acid (ATRA), a potentactivator of RARα signaling. ATRA supplementation of mouse fibroblastsdid not affect total rates of protein degradation under basalconditions, but significantly reduced the increase in proteindegradation normally observed in these cells in response to prolongedserum removal (FIG. 3a ). Addition of inhibitors of lysosomalproteolysis confirmed that both basal and inducible lysosomaldegradation were significantly compromised after ATRA supplementation(FIG. 3b ). In contrast to the reports in other cell types^(25,27,28),there were no significant changes in steady state levels of LC3 or inits lysosomal flux analyzed by immunoblot (FIG. 3c ) or with the moredynamic mcherry-GFP-LC3 reporter, supporting the previously proposedcell-type dependent stimulatory effect of retinoids on macroautophagy.Analysis of CMA using the CMA reporter revealed that treatment with ATRAdid not have a noticeable effect on basal CMA but significantly reducedthe activation of this pathway in response to serum removal. Overallthese findings support that ATRA and RARα signaling exert an inhibitoryeffect on CMA activity.

Design and synthesis of RARα antagonist with selective effect on CMA.Previous reports have revealed that the effect of ATRA on macroautophagywas not exerted through RAR signaling, as it was independent of thepresence or absence of these reeptors²⁸. Signaling through RARα was notbehind the observed inhibitory effect on autophagic degradation ofcytosolic proteins, because it was still detectable when RARα (−) cellswere supplemented with ATRA. In contrast, the inhibitory effect of ATRAon CMA was dependent on the RARα, as ATRA treatment no longer inhibitedCMA activity in RARα (−) cells. The marked upregulation of CMA observedwhen RARα was eliminated, the opposite effects of this intervention onmacroautophagy activity, and the fact that part of the effect of ATRA onautophagy was not mediated through RARα signaling led to the presentproposal that it may be possible to design RARα-targeted compoundscapable of upregulating CMA activity without affecting other autophagicpathways. Furthermore, since RARα has the capability to activate andrepress target-gene transcription, and the molecular determinants ofthis repression have been recently identified³¹, the aim was to identifyantagonist molecules selective for this repressor activity of RARα. Tothis effect, structure-based chemical design strategies and novelchemistry were used to generate a small library of retinoic acidderivatives.

Using the structure of ATRA, chemical changes were introduced to protectthe regions of this molecule most prone to intracellular modificationsand to enhance ATRA reactive properties with RARα. FIG. 4a depicts thethree basic domains common to all retinoid molecules: a hydrophobiccomponent, an all-trans-configured alkene linker, and a polar group (thecarboxylic acid moiety)²². The alkene linker region is sensitive tophotochemical changes²¹ and to oxidation at the allylic C4 position inthe trimethylcyclohexyl ring by enzymes such as cellular isomerases andcytochrome P450²².

Through chemical modifications using structure-based chemical designapproach, a library of 29 retinoic acid-related compounds was generatedthat was grouped in four different families: α-aminonitrile retinoids(αAmR), boron-aminonitrile retinoids (BAmR), guanidine retinoids (GR),and atypical retinoids (AR) protected in specific positions (FIG. 4a )².These families contain modifications at the C4 position of thehydrophobic ring, to protect it from possible oxidation to 4-oxo, and ingeneral shortened alkene linkers, which are susceptible tophotoisomerization (FIG. 4a ; the portions of the molecules where thecompounds differ are highlighted and the conserved typical retinoidregions are highlighted). The new designed alkene linker conserves itsaromaticity in the ring-constrained form. In addition, groups such as—CN, —NH or the boron atom were incorporated with the aim of enhancingATRA-reactive properties (for example, sp/sp³ hybridization of the borongroup can facilitate formation of hydrogen or covalent bonds). Thestructures of all the compounds generated for the library and theirsynthetic schemes are shown in Table 1.

Before analyzing the effect of the library compounds on CMA, the effectof increasing concentrations of each of them on cellular viability wasdetermined. Except for four compounds that showed toxic effects at allconcentrations tested (data not shown), for most compounds toxicity wasnot clearly manifested until concentrations ≥50 μM. Consequently, forall subsequent testing, compounds were used at 20 μM, which resulted inless than 20% decrease in cellular viability and no evidence ofapoptosis induction in using annexin V labeling in serum deprived cells.The effect of all the compounds in the library on CMA was screened usingmouse fibroblasts expressing the CMA reporter. For those compoundsshowing a positive effect with the CMA reporter (increase in the numberof fluorescent puncta in cells maintained in the presence of serumhigher than 2.5-fold), changes in total protein degradation werevalidated using metabolic labeling. This combined analyses identifiedmarked activation of CMA activity in cells treated with compounds AR7,GR1, and GR2 in a dose-dependent manner. The schemes of the syntheticreactions for the generation of these three compounds are shown in FIG.4b ³². NMR data show that GR1 compound had a mixture of isomers with E-and Z-stereoselectivity in 2:1 ratio whereas in GR2 the major isomer wasE and the minor Z in a 1:0.2 ratio.

To determine the effect of these molecules on RAR signaling, cells wereco-transfected cells with a plasmid coding for RAR fused to the Gal4DNA-binding domain and a Gal4-dependent luciferase reporter. In contrastto the dose-dependent activation observed in cells treated with ATRA,similar doses of the three compounds that activate CMA did not have anyeffect on luciferase activity (FIG. 5a ), whereas some compounds in theother families in the library (αAmR and BAmR) displayed discreteactivity. When administered in combination with ATRA, these compoundshad a marked inhibitory effect on the ATRA-dependent activation ofluciferase (FIG. 5b ). In fact, GR2 and AR7 were among the most potentantagonist compounds in the library. Using a similar luciferase-basedreporter for RXR, although some compounds in the library (αAmR family)exhibited activity through this receptor, none of the three leadingcompounds had significant agonist or antagonist activity on thisreceptor (FIG. 5c,d ).

The RAR antagonistic potency of the three leading molecules was close tothat of the bona fide antagonist BMS614 (FIG. 5b ). In fact, BMS614 alsoenhanced CMA activity, but whereas this antagonist inhibitedmacroautophagy, none of the compounds significantly affectedautophagosome content or their clearance by lysosomes (FIG. 5e ). Theseresults confirm that the novel retinoid derivatives act as RARαantagonists and are capable of upregulating CMA without affectingmacroautophagy.

Stimulatory effect of the novel retinoid derivatives on CMA. The effecton autophagy of the three novel RARα antagonist compounds generated inthis study was further characterized. Using RARα knock-down cells, thestimulatory effect of the new derivatives on total protein degradation(FIG. 6a ) and on CMA (FIG. 6b ) was confirmed to be dependent on thepresence of RARα. Thus, although both activities were higher in the RARα(−) cells, addition of the retinoid derivatives no longer had astimulatory effect. The increase in protein degradation (FIG. 6a ) andin the amount of CMA-positive puncta per cell (FIG. 6b ) induced by theretinoid derivates was also confirmed to be a result of activation ofCMA, as their effect was abolished in cells knocked down for thelysosomal CMA receptor, and consequently incapable of carrying on CMA³³.In contrast, these compounds were still capable of effectivelyactivating CMA in cells knocked-down for LAMP-2B, a protein with 85%homology to LAMP-2A but that does not participate in CMA (FIG. 6c ).

To start elucidating the basis for the differences in the antagonisticeffect on RARα activity of the new retinoid derivates when compared withother well characterized antagonist molecules, molecular docking andmolecular dynamics simulations were performed with the three leadingcompounds. Molecular docking studies of the compounds were performedwith the RARα X-ray crystal structure in the inactive coformation³¹.Docking studies revealed that the three leading compounds may bind withtwo different orientations (pose I and pose II) in the ligand-bindingsite of the RA receptor. Both docking orientations demonstrate that AR7,GR1 or GR2 do not interact or are in close distance with the catalyticArg272, a feature that is common to ATRA and other known agonists andantagonists. The binding site in docking pose I is formed by residues ofhelices H3, H10 and H12 and in pose II by residues of helices H3, H10and H5. As a result, the compounds adopt opposite orientation in poses Iand II and have a small overlap of interacting residues. Docking alsorevealed that both E- and Z-isomers of GR1 and GR2 can bind with the twodifferent poses I and II and that E- and Z-isomerization have a minimalimpact on the docked structure of these compounds suggesting that bothisomers can be active RARα antagonists.

Analysis of docking and molecular dynamics simulations favorablysuggests that the compounds adopt a binding mode, as shown in FIGS. 4cand d . In this docking orientation, the compounds are placed at thejunction of α-helices H12, H3 and H10. This is an interesting dockingorientation that is consistent with the functional activity of thecompounds and mimics a portion of the known antagonist BMS614 when isbound to RARα¹³. The compounds form extensive hydrophobic interactionswith the hydrophobic residues of the site suggestingstabilizing-interactions with the open conformation of the α-helix H12that regulates recruitment of RARα co-regulators. Compounds would havesteric clashes with the α-helix H12 in the closed conformation when RARαis in active mode. Additionally, docking suggests that the guanidiniumgroup of compounds GR1 and GR2 can form hydrogen bonds with the carbonylbackbone of Pro407 in α-helix H12 and the hydroxyl group of Thr233 inα-helix H3. Thus, docking positions the compounds in a critical regionof the RARα binding site to stabilize its inactive conformation. Thisbinding region is geographically distinct from the ATRA binding site andmay account for the selectivity of the compounds for RARα.

The result of the two possible docking orientations can be explained bythe small size of the lead molecules compared to the RARα binding siteand the high complementarity of hydrophobic interactions between thecompounds and the RARα residues in the two docking orientations. It washypothesized that it would be possible that AR7 and the GR compoundscould bind simultaneously to the RARα binding site. To experimentallytest this possibility, the effect on CMA of AR7 and GR1 added alone orin combination was analyzed. A marked increase in their CMA activatingpotency was found when the same final concentration was reached bycombining both molecules, supporting a co-operative effect of these twocompounds when added together. Thus, the potential ability of the AR7and GR compounds to bind in two orientations, that are still differentfrom the bound pose of ATRA and other known RARα modulators, along withthe complexity of ligand-induced changes already described for thisreceptor—that affect RARα-DNA binding but also modulatedissociation/association of several co-regulatory complexes—couldcontribute to the different downstream consequences of theirantagonistic effect on RARα.

FIG. 8 illustrates the specificity of compounds GR1, GR2 and AR7 towardCMA and the absence of effect on other autophagic pathways(macroautophagy shown here). Contrary to the commercially availableretinoic acid antagonists that have a profound inhibitory effect onmacroautophagy, it is shown here by measuring LC3 flux that the presentcompounds do not inhibit macroautophagy.

Protective effect of the novel CMA activators against proteotoxicity. Togain insights on the mechanism by which the new retinoid derivatesactivate CMA, their effect on the cellular oxidative status wasanalyzed. Retinoic acid has been shown to exert anti-oxidant effects ina variety of cellular settings³⁴. It is thus plausible that inhibitionof RARα signaling could result in enhanced oxidative stress, a conditionknown to activate CMA³⁵. Immunoblot with an antibody againstcarbonylated groups to detect oxidized proteins in cell lysates revealedno differences between cells untreated or after treatment with thedifferent retinoid derivatives. These results make it unlikely thatactivation of CMA in the treated cells was reactive to an increase inoxidative stress on those cells. It was also analyzed whether thecompounds may exert their stimulatory effect on CMA by directly actingon the lysosomal compartment. Pretreatment of intact lysosomes isolatedfrom rat liver with the different retinoid derivates did not increasebinding or uptake of the well-characterized CMA substrateglyceraldehyde-3-phosphate dehydrogenase (GADPH) in these lysosomes whensubjected to a standard in vitro assay for CMA (FIG. 7a ). In contrastto this lack of lysosomal effect in vitro, analysis of lysosomesisolated from cells treated with the different retinoid derivativesrevealed a higher content of the endogenous substrate GAPDH in thiscompartment, supportive of enhanced CMA and further supporting that thecompounds activate CMA through RARα signaling and not directly byinteracting with CMA components in lysosomes (FIG. 7b ).

No significant differences were observed on the ability of RARα totranslocate to the nucleus in response to ATRA in the presence of theretinoic derivatives disproving a possible inhibitory effect at thislevel. In fact, addition of AR7 was sufficient to stimulate somerelocation of cytosolic RARα to the nucleus. Treatment with thetranscriptional repressor Actinomycin D partially reduced thestimulatory effect of AR7 on CMA, supporting contribution oftranscriptional changes to the upregulation of CMA.

In the search for CMA targets modulated by the retinoid derivatives, thelysosomal receptor LAMP-2A was focused on, as its levels are limitingfor CMA. Higher levels of this receptor were found in the lysosomes fromthe treated cells (FIG. 7b ), along with a discrete but significanttranscriptional activation of LAMP-2A in these cells, comparable to thatpreviously described in conditions of maximal activation of CMA such asparaquat-induced oxidative stress (FIG. 7c ). This transcriptionalupregulation was not observed for other lysosomal membrane proteins.LAMP-1 was partially inhibited by treatment with Actinomycin D and wasno longer observed in the absence of the RARα (FIG. 7c ). No changes inLAMP-2A mRNA levels were found upon stimulation of RARα activity withATRA under basal conditions, in agreement with the lack of effect ofATRA treatment on basal CMA. However, a significant decrease in LAMP-2AmRNA was found when ATRA was added to cells deprived of serum, acondition in which ATRA treatment reduced CMA activity.

Lastly, the possible beneficial effect of chemical enhancement of CMAwith the new retinoid derivatives in cellular homeostasis and itsresistance to stress were investigated. To this end, a comparison wasmade of the sensitivity to the pro-oxidant compound paraquat (PQ) ofcells treated with the retinoid derivatives before or right after theoxidative insult. As shown in FIG. 7d , addition of retinoid derivativesto cells exposed to PQ for 4 hours had only a very discrete positiveeffect on cellular viability. In contrast, when the compounds were addedbefore inducing oxidative stress, a marked improvement in cellularviability was observed (FIG. 7d , top). The enhanced resistance to theoxidative insult in retinoid-treated cells was mainly due to theirstimulatory effect on CMA, because their protective effect wascompletely abolished in cells unable to carry out CMA (knocked-down forLAMP-2A) (FIG. 7d , bottom). Note that the high sensitivity ofCMA-incompetent cells to oxidative stress required use in these cells ofone-fourth of the concentration of PQ used in control cells.

Maintained chronic oxidation is a common feature of aging and anaggravating factor in multiple degenerative disorders. To model thedetrimental effect of oxidative stress on proteotoxicity and analyze thepossible beneficial effect of enhancing CMA activity in theseconditions, cells were transfected with α-synuclein, the protein thataccumulates in the form of intracellular aggregates in Parkinson'sdisease (PD) and previously identified as a bonafide CMAsubstrate^(9,10). Although mutations in this protein have beenassociated to the familial forms of the disease, in more than 98% of PDpatients the protein that accumulates in the protein inclusions is wildtype. Consequently, different intracellular factors and aggressors,including oxidative stress, have been proposed to contribute to theidiopathic forms of this pathology. In the present experimentalconditions, transfection of cultured cells with increasingconcentrations of cDNA coding for α-synuclein did not result in toxicityuntil concentrations above 10 μg were used (FIG. 7e ). Although thepresence of α-synuclein alone was not toxic for these cells, thetoxicity of a fixed concentration of PQ was clearly dependent on theconcentration of this protein present in the cell. The concentration ofPQ added to these cells was adjusted so that in cells expressing lowlevels of α-synuclein, its toxic effect was limited to no more than 20%reduction in cellular viability. However, the same concentrations of PQresulted in reductions of up to 80% in cellular viability in cellsexpressing higher concentrations of α-synuclein (FIG. 7e ). The toxiceffect in the experimental paradigm resulted from the combination of theoxidative stress and the proteotoxicity associated to α-synuclein,rather than to DNA toxicity, because the effect of PQ remained constantin cells transfected with increasing concentrations of an empty plasmid.Activation of CMA, by pre-treatment of α-synuclein-transfected cellswith the novel retinoid derivatives (compound AR7 shown in FIG. 7e )before addition of PQ, resulted in significant increases in cellularviability, even in cells expressing very high levels of α-synuclein.Pre-treatment with the compounds also reduced the formation ofoligomeric species of α-synuclein observed in the cells expressingα-synuclein and treated with PQ (FIG. 7f ).

These results support the beneficial effect that upregulation of CMA hasin the cellular defense against oxidative stress and proteotoxicity andconfirm the efficacy of the novel retinoid derivatives to activate CMAeven under pathological conditions.

Discussion

The recently gained appreciation for the importance of selective formsof autophagy, such as CMA, in the maintenance of cellular homeostasisand the contribution of their malfunctioning to human disease, hasresulted in a growing interest in development of chemical modulators ofthese pathways. Despite recent findings supporting a direct compromiseof CMA in neurodegenerative diseases, diabetes and lysosomal storagedisorders^(9,11,12) and the pronounced beneficial effect observed whenthe age-dependent decline of this pathway is prevented¹⁵, chemicalmodulators of CMA were for the most part lacking until now. One of themain limitations for the development of CMA activators and inhibitorshas been the absence of chemical targets for this pathway. Most of thekey components for this pathway are multifunctional proteins thatparticipate in many other cellular processes, which would make theirchemical targeting for modulation of CMA very nonspecific. LAMP-2A, themost unique component for CMA, is also a difficult target due to itshigh homology (almost 85% identity) with the other spliced variants ofthe lamp2 gene, known to participate in other cellular functions such asmacroautophagy, lysosomal biogenesis and cholesterol trafficking³⁶.

The present work identified a novel regulator of CMA activity amenablefor chemical targeting. Signaling through the RAR receptor was found toexert an inhibitory effect on CMA. Although complete disruption ofsignaling through this receptor by knock-down of the receptor protein iseffective in attaining maximal CMA activation, this intervention leadsto inhibition of macroautophagy. Using structure-based chemical design,it was possible to dissociate the opposite effects of RARα onmacroautophagy and CMA and to generate compounds capable of antagonizingonly the RARα inhibitory effect on CMA, without affectingmacroautophagy. The protective effect of the upregulation of CMAmediated by these compounds against oxidative stress and proteotoxicitywere also demonstrated.

The high antagonistic effect of the novel retinoid derivatives likelyresults from the combination of tight binding to RARα (favored by themultiplicity of contact sites within the binding pocket) and highstability (obtained by protecting the sites in retinoids usuallyamenable to intracellular modification). For example, in GR1 and GR2,the polyene linker and the hydrophobic ring are predominantly surroundedby hydrophobic side chains that protect this region, and the interactionwith RARα residues Thr233, Ser229 and Pro407 may contribute to theresonance stabilization of the guanidine type polar moiety (FIGS. 4d and4f ). Similarly, in the case of AR7, the bulky aromatic rings offerexcellent interaction contacts with the surrounding hydrophobic RARαresidues such as Met406, Leu266, Leu398 and Ile270 (FIG. 4c ).

Reciprocal cross-talk between macroautophagy and CMA has been previouslyreported and it is behind the compensatory activation of one of thesepathways when the other malfunctions⁵. The present findings support thatRARα signaling could be one of the mechanisms that modulate thecross-talk between both autophagic pathways, judging by its oppositeeffect on them. In practice, implementation of treatments aimed atupregulating CMA by targeting RAR signaling requires dissociating thiseffect from the one observed on macroautophagy, as otherwiseupregulation of CMA will lead to undesired reduction in macroautophagicactivity. Both gene activation and gene repression have been describedto occur through the complex family of RARs, and examples of redundantand type-specific functions for each of the members of this family havebeen reported¹⁷. This functional diversity suggests that the interactionof RAR molecules with their targets is probably modulated by multiplefactors, including unique characteristics of the binding of ligands tothis receptor. This last property and been exploited to introducechemical modifications in the ligands to favor the effect of RARα on aparticular subset of targets that in turn leads to the selectiveactivation of CMA.

Activation of CMA by the novel retinoid derivatives generated in thisstudy is not reactive to blockage of macroautophagy or to a higheroxidative intracellular environment. Instead, upregulation of CMA occursselectively, and in contrast to the receptor-independent effectsdescribed for ATRA on macroautophagy, it depends on a functional RARα.Evidence is presented that LAMP-2A is one of the downstream targets ofthis pathway. The fact that the LAMP-2A gene does not contain arecognizable retinoic acid response element region and that the retinoidderivatives suppressed rather than activate a reporter with thatsequence, suggest that transcriptional activation of LAMP-2A is undernegative control by RARα. It is noteworthy to point out that althoughmost lysosomal proteins are under a common transcriptional programcontrolled by TFEB³⁷, LAMP-2A is one of the few exceptions. RARαsignaling is thus the first signaling mechanism shown to regulate thislysosomal receptor. Interestingly, ChIP-Seq analysis have revealed thatone of the two largest functional classes of RAR target genes wasrelated to proteolysis³⁸. The present study contributes to furtherreinforce the importance of RAR signaling in protein degradation.

The protective effect against oxidation and proteotoxicity observed ofthe new retinoid derivatives supports the therapeutic potential of theseand related compounds in chronic age-related diseases. Maintenance ofprotein homeostasis is achieved through a tightly coordinated balancebetween chaperones and proteolytic systems. It is essential thus, todevelop interventions that can separately affect one of these processeswithout compromising the functionality of the other cellular qualitycontrol mechanisms. The efficient upregulation of CMA observed with theretinoid derivatives and their lack of noticeable effects onmacroautophagy makes them suitable for the selective modulation of CMAin those conditions in which this pathway is primarily compromised suchas neurodegeneration and in aging.

TABLE 1 Synthesis and properties of the four groups of retinoidderivatives generated for this study Atypical Retinoids

C1 AR1: R₁ = R₂ = H; R₃ = Cl AR2: R₁ = NO₂; R₂ = R₃ = H AR3: R₁ = H; R₂= F; R₃ = Cl AR4: R₁ = R₃ = Cl; R₂ = H AR5: R₁ = H; R₂ = CH₃; R₃ = FAR6: R₁ = R₃ = Cl; R₂ = CH₃ AR7: R₁ = Cl; R₂ = H; R₃ = CH₃ AR8: R₁ = R₃= H; R₂ = Cl AR9: R₁ = R₂ = R₃ = H AR10: R₁ = R₃ = H; R₂ = NO₂ NMR (Ref33) Guanidine Retinoids

C2 GR1: R₁ = R₂ = CH₃ C3 GR2: R₁ = R₂ = H NMR (Ref 32) α-AminonitrileRetinoids

αAmR1: R₁ = R₂ = R₃ = H αAmR2: R₁ = NO₂; R₂ = R₃ = H αAmR3: R₁ = F; R₂ =R₃ = H αAmR4: R₁ = I; R₂ = R₃ = H αAmR5: R₁ = H; R₂ = OH; R₃ = NO₂αAmR6: R₁ = OH; R₂ = R₃ = H αAmR7: R₁ = CH₂—CH₂—OH; R₂ = R₃ = H αAmR8:R₁ = OCH₃; R₂ = R₃ = H αAmR9: R₁ = Cl; R₂ = R₃ = H αAmR10: R₁ = methylcarboxylate; R₂ = R₃ = H αAmR11: R₁ = Boronate ester; R₂ = R₃ = H NMR(Ref 31) Boron-Aminonitrile Retinoids

C4 BAmR1: = Ar = Ph C5 BAmR2: = Ar = 4-F—Ph

C6 BAmR3: HNR₁R₂ = py; BR₃R₄ = Boronate ester C7 BAmR4: HNR₁R₂ = an;BR₃R₄ = Boronate ester C8 BAmR5: HNR₁R₂ = dba; BR₃R₄ = Boronate ester C9BAmR6: HNR₁R₂ = pi; BR₃R₄ = Boronate ester HNR₁R₂ = pyrrolidine (py); =aniline (an); = dibenzyl amine (dba); = piperidine (pi);

Example B Overview

Chaperone-mediated autophagy (CMA) contributes to cellular qualitycontrol and the cellular response to stress through the selectivedegradation of cytosolic proteins in lysosomes. Pathogenic proteins ofcommon neurodegenerative disorders such as Parkinson's and Huntington'sdisease or frontotemporal dementia have been shown to undergodegradation via CMA. A decrease in CMA occurs in aging and therefore maycontribute to accelerate the course of age related disorders. There arevery limited options for the chemical modulation of CMA. CMA isinhibited by signaling from the nuclear retinoic acid receptor α (RARα).In Example A, RARα antagonists (AR7, GR1 and GR2) were described thatcan selectively activate CMA without affecting other cellular clearancepathways. A structure-based drug design and medicinal chemistry, basedon the AR7 scaffold, have now been applied to increase the CMAactivation potency. Using a photoactivatable fluorescent CMA reporter,the CMA activation potency of these new compounds was determined incultured cells. Compared with AR7, most of the new compounds showedsimilar to better activity. Compound QX39 showed 2-3 fold higher potencythan AR7, and in contrast to the parent molecule that only activatedbasal CMA. Several compounds including compound QX39 activated bothbasal and stress-induced CMA. The effect of these compounds in proteindegradation has been demonstrated using metabolic labeling in culturedcells. Several of the new compounds demonstrated better stimulatoryeffect on lysosomal degradation and protective effect on cell viabilityupon induction of oxidative stress, proteotoxicity and lipotoxicity inmouse embryo fibroblasts or neuronal cell lines.

Design and Synthesis of Example Compounds Example 1:3-([1,1′-biphenyl]-4-yl)-7-chloro-2H-benzo[b][1,4]oxazine (A-1)

To 2-amino-5-chlorophenol (143.6 mg, 1 mmol) in acetonitrile (10 mL) wasadded K₂CO₃ (0.27 g, 2 mmol). Into this,1-([1,1′-biphenyl]-4-yl)-2-bromoethan-1-one (280 mg, 1.1 mmol) inacetonitrile (15 mL) was added dropwise at room temperature. Thereaction was then stirred overnight under reflux. Then the solvent wasevaporated and the residue was dissolved in dichloromethane (20 mL). Theorganic layer was washed with water, brine and dried over Na₂SO₄. Thedesired compound was isolated through silica gel chromatography.Recrystallization with hot ethanol gave a light yellow powder (A-1, 80mg, 25%). MS (ESI) M+H⁺=320.09.

Example 2: 3-([1,1′-biphenyl]-4-yl)-7-methyl-2H-benzo[b][1,4]oxazine(A-2)

To 2-amino-5-methylphenol (123.2 mg, 1 mmol) in dichloromethane (10 mL)was added K₂C3 (0.27 g, 2 mmol), tetrabutylammonium bisulfate (17 mg,0.05 mmol). Into this, 1-([1,1′-biphenyl]-4-yl)-2-bromoethan-1-one (280mg, 1.1 mmol) in dichloromethane (15 mL) was added dropwise at roomtemperature. The reaction was then stirred overnight under reflux. Thereaction was washed with water, brine and dried over Na₂SO₄. The desiredcompound was isolated through silica gel chromatography.Recrystallization with hot ethanol gave a light yellow powder (A-2, 57mg, 19%). MS (ESI) M+H⁺=300.13.

Example 3:3-([1,1′-biphenyl]-4-yl)-7-(trifluoromethyl)-2H-benzo[b][1,4]oxazine(A-3)

Following a procedure analogous to the procedure described in example 2using 2-amino-5-(trifluoromethyl)phenol (97.4 mg, 0.55 mmol) and1-Q1,1′-biphenyl-4-yl)-2-bromoethan-1-one (137.5 mg, 0.5 mmol). Thedesired compound was obtained as white solid (A-3, 41.5 mg, 24.5%). MS(ESI) M+H⁺=354.24.

Example 4:3-([1,1′-biphenyl]-4-yl)-7-(trifluoromethyl)-2H-benzo[b][1,4]oxazine(A-4)

Following a procedure analogous to the procedure described in example 2using 2-amino-5-fluorophenol (69.9 mg, 0.55 mmol) and1-([1,1′-biphenyl]-4-yl)-2-bromoethan-1-one (137.5 mg, 0.5 mmol). Thedesired compound was obtained as white solid (A-4, 39 mg, 25.7%). MS(ESI) M+H⁺=304.16.

Example 5: 7-chloro-3-(4-(pyridin-4-yl)phenyl)-2H-benzo[b][1,4]oxazine(A-5): (QX 136) Step 1:3-(4-bromophenyl)-7-chloro-2H-benzo[b][1,4]oxazine (Intermediate 1)

Following a procedure analogous to the procedure described in example 2using 2-amino-5-chlorophenol (287.14 mg, 2 mmol) and2-bromo-1-(4-bromophenyl)ethan-1-one (555.9 mg, 2 mmol). The desiredcompound was obtained as white solid (intermediate 1, 333 mg, 51.6%). MS(ESI) M+H⁺=322.95.

Step 2: 7-chloro-3-(4-(pyridin-4-yl)phenyl)-2H-benzo[b][1,4]oxazine(A-5)

A nitrogen flushed vessel was filled with3-(4-bromophenyl)-7-chloro-2H-benzo[b][1,4]oxazine (intermediate 1, 50mg, 0.15 mmol), pyridin-4-ylboronic acid (22.7 mg, 0.185 mmol) andCs₂CO₃ (97.7 mg, 0.3 mmol, 2N in water). The vessel was flushed withnitrogen for a second time and Tetrakis(triphenylphosphine)palladium(0)(17.3 mg, 0.015 mmol) was added. Then solvent dioxane (15 mL) was addedand the reaction was degassed and protected with nitrogen, and stirredat 80° C. overnight. Then the solvent was evaporated and the residue wasdissolved in dichloromethane (25 mL). The organic layer was washed withwater, brine and dried over Na₂SO₄. The desired compound was isolatedthrough silica gel chromatography. Recrystallization with hot ethanolgave a light yellow powder (A-5, 43 mg, 89%). MS (ESI) M+H⁺=320.07.

Example 6:7-chloro-3-(4-(isoquinolin-4-yl)phenyl)-2H-benzo[b][1,4]oxazine (A-6)

Following a procedure analogous to the procedure described in step 2 ofexample 5, using 3-(4-bromophenyl)-7-chloro-2H-benzo[b][1,4]oxazine(intermediate 1, 100 mg, 0.3 mmol) and isoquinolin-4-ylboronic acid(62.3 mg 0.36 mmol). The desired compound was obtained as off-whitepowder (A-6, 81.3 mg, 73.1%). MS (ESI) M+H⁺=371.13.

Example 7:1-(3-(4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)phenyl)thiophen-2-yl)ethan-1-one(A-7)

Following a procedure analogous to the procedure described in step 2 ofexample 5, using 3-(4-bromophenyl)-7-chloro-2H-benzo[b][1,4]oxazine(intermediate 1, 100 mg, 0.3 mmol) and (2-aceylthiophen-3-yl)boronicacid (61.2 mg, 0.36 mmol). The desired compound was obtained asoff-white powder (A-7, 68.2 mg, 61.8%). MS (ESI) M+H⁺=368.43.

Example 8: methyl4′-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)-6-methyl-[1,1′-biphenyl]-3-carboxylate(A-8)

Step 1:7-chloro-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2H-benzo[b][1,4]oxazine(intermediate 2): A nitrogen flushed vessel was filled with3-(4-bromophenyl)-7-chloro-2H-benzo[b][1,4]oxazine (intermediate 1, 800mg, 2.48 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (756 mg,2.98 mmol) and potassium acetate (730.2 mg, 7.44 mmol). The vessel wasflushed with nitrogen for second time and[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (204.16 mg,0.25 mmol) was added. Then solvent dimethyl sulfoxide (50 ml) was addedand the reaction was degassed and protected with nitrogen, and stirredat 80° C. for 2 hours. Then the reaction solution was filtered and thefiltrate was diluted with acetate ester (200 mL). The organic layer waswashed with water, brine and dried over Na₂SO₄. The desired compound wasisolated through silica gel chromatography to give a pink powder(intermediate 2, 500 mg, 54.5%). MS (ESI) M+H⁺=370.14.

Step 2: methyl4′-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)-6-methyl-[1,1′-biphenyl]-3-carboxylate(A-8): Following a procedure analogous to the procedure described instep 2 of example 5, using7-chloro-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2H-benzo[b][1,4]oxazine(intermediate 2, 120 mg, 0.32 mmol) and methyl 3-bromo-4-methylbenzoate(89.2 mg, 0.39 mmol). The desired compound was obtained as white powder(A-8, 104 mg, 82.9%). MS (ESI) M+H⁺=392.30.

Example 9:7-chloro-3-(4′-chloro-[1,1′-biphenyl]-4-yl)-2H-benzo[b][1,4]oxazine(A-9)

Following a procedure analogous to the procedure described in step 2 ofexample 5, using 3-(4-bromophenyl)-7-chloro-2H-benzo[b][1,4]oxazine(intermediate 1, 100 mg, 0.3 mmol) and (4-chlorophenyl)boronic acid(36.3 mg, 0.36 mmol) in water/ethanol/toluene (1.5/2.5/10, 14 mL). Thedesired compound was obtained as white powder (A-9, 30 mg, 28.2%). MS(ESI) M+H⁺=354.04.

Example 10: 4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)benzonitrile (A-10)

Following a procedure analogous to the procedure described in example 2,using 2-amino-5-chlorophenol (143.6 mg, 1 mmol) and4-(2-bromoacetyl)benzonitrile (246.5 mg, 1.1 mmol). The desired compoundwas obtained as white powder (A-10, 42 mg, 16%). MS (ESI) M+H⁺=269.31.

Example 11: 4-(7-methyl-2H-benzo[b][1,4]oxazin-3-yl)benzonitrile (A-11)

Following a procedure analogous to the procedure described in example 2,using 2-amino-5-methylphenol (123.2 mg, 1 mmol) and4-(2-bromoacetyl)benzonitrile (246.5 mg, 1.1 mmol). The desired compoundwas obtained as yellow crystal (A-11, 108 mg, 43.5%). MS (ESI)M+H⁺=249.14.

Example 12: 4-(7-fluoro-2H-benzo[b][1,4]oxazin-3-yl)benzonitrile (A-12)

Following a procedure analogous to the procedure described in example 2,using 2-amino-5-fluorophenol (168 mg, 0.75 mmol) and4-(2-bromoacetyl)benzonitrile (104.9 mg, 0.825 mmol). The desiredcompound was obtained (A-12, 81 mg, 42.8%). MS (ESI) M+H⁺=253.06.

Example 13:4-(7-(trifluoromethyl)-2H-benzo[b][1,4]oxazin-3-yl)benzonitrile (A-13)

Following a procedure analogous to the procedure described in example 2,using 2-amino-5-(trifluoromethyl)phenol (97.4 mg, 0.55 mmol) and1-([1,1′-biphenyl]-4-yl)-2-bromoethan-1-one (137.5 mg, 0.5 mmol). Thedesired compound was obtained as white solid (A-13, 128 mg, 56.5%). MS(ESI) M+H⁺=303.09.

Example 14:7-methyl-3-(4-(trifluoromethyl)phenyl)-2H-benzo[b][1,4]oxazine (A-14)

Following a procedure analogous to the procedure described in example 2,using 2-amino-5-methylphenol (123.2 mg, 1 mmol) and2-bromo-1-(4-(trifluoromethyl)phenyl)ethan-1-one (320.5 mg, 1.2 mmol).The desired compound was obtained as flake solid (A-14, 136 mg, 46.6%).MS (ESI) M+H⁺=292.08.

Example 15:7-chloro-3-(4-(trifluoromethyl)phenyl)-2H-benzo[b][1,4]oxazine (A-15)

Following a procedure analogous to the procedure described in example 2,using 2-amino-5-chlorophenol (143.6 mg, 1 mmol) and2-bromo-1-(4-(trifluoromethyl)phenyl)ethan-1-one (293.7 mg, 1.1 mmol).The desired compound was obtained as flake solid (A-15, 103 mg, 48.6%).MS (ESI) M+H⁺=312.23.

Example 16:7-methyl-3-(4-(trifluoromethoxy)phenyl)-2H-benzo[b][1,4]oxazine (A-16)

Following a procedure analogous to the procedure described in example 2,using 2-amino-5-methylphenol (61.6 mg, 0.5 mmol) and2-bromo-1-(4-(trifluoromethoxy)phenyl)ethan-1-one (155.6 mg, 0.6 mmol).The desired compound was obtained as flake solid (A-16, 40 mg, 26.1%).MS (ESI) M+H⁺=308.08.

Example 17: methyl 4-(7-methyl-2H-benzo[b][1,4]oxazin-3-yl)benzoate(A-17)

Following a procedure analogous to the procedure described in example 2,using 2-amino-5-methylphenol (47 mg, 0.38 mmol) and methyl4-(2-bromoacetyl)benzoate (108 mg, 0.42 mmol). The desired compound wasobtained as flake solid (A-17, 74.1 mg, 69%). MS (ESI) M+H⁺=282.23.

Example 18: 4-(7-methyl-2H-benzo[b][1,4]oxazin-3-yl)benzoic Acid (A-18)

A-17 (35 mg, 0.124 mmol) was dissolved in THF (4 mL). 1 M LiOH (1.24mmol, 1.24 mL) was added and the resulting mixture was stirred at roomtemperature overnight. Then dichloromethane (15 mL) was added to thereaction. 1N HCl (5 mL) was added and stirred for 5 minutes. The organiclayer was washed with brine and dried over Na₂SO₄. The desired compoundwas isolated through silica gel chromatography to afford yellow powder(A-18, 23 mg, 69%). MS (ESI) (M−H)⁻=266.46.

Example 19: methyl 4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)benzoate(A-19)

Following a procedure analogous to the procedure described in example 2,using 2-amino-5-chlorophenol (25.8 mg, 0.18 mmol) and methyl4-(2-bromoacetyl)benzoate (41.1 mg, 0.16 mmol). The desired compound wasobtained as white solid (A-19, 31 mg, 64.2%). MS (ESI) M+H⁺=302.57.

Example 20: N-(4-(7-methyl-2H-benzo[b][1,4]oxazin-3-yl)phenyl)acetamide(A-20)

Following a procedure analogous to the procedure described in example 1,using 2-amino-5-methylphenol (61.58 mg, 0.5 mmol) andN-(4-(2-bromoacetyl)phenyl)acetamide (154 mg, 0.6 mmol). The desiredcompound was obtained as white solid (A-20, 33 mg, 24%). MS (ESI)M+H⁺=281.24.

Example 21: N-(4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)phenyl)acetamide(A-21)

Following a procedure analogous to the procedure described in example 1,using 2-amino-5-chlorophenol (71.7 mg, 0.5 mmol) andN-(4-(2-bromoacetyl)phenyl)acetamide (154 mg, 0.6 mmol). The desiredcompound was obtained as white solid (A-21, 46 mg, 30.6%). MS (ESI)M+H⁺=301.57.

Example 22: N-(4-(7-methyl-2H-benzo[b][1,4]oxazin-3-yl)phenyl)acetamide(A-22)

Following a procedure analogous to the procedure described in example 1,using 2-amino-5-(trifluoromethyl)phenol (88.6 mg, 0.55 mmol) andN-(4-(2-bromoacetyl)phenyl)acetamide (154 mg, 0.6 mmol). The desiredcompound was obtained as white solid (A-22, 75.5 mg, 22.6%). MS (ESI)M+H⁺=335.38.

Example 23: 4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)benzoic Acid (A-23)

Step 1: 2-(trimethylsilyl)ethyl4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)benzoate (intermediate 3):Following a procedure analogous to the procedure described in example 2,using 2-amino-5-chlorophenol (71.8 mg, 0.5 mmol) and2-(trimethylsilyl)ethyl 4-(2-bromoacetyl)benzoate (WO 2009112615) (188mg, 0.55 mmol). The desired compound was obtained as white powder(intermediate 3,130 mg, 67%). MS (ESI) M+H⁺=388.78.

Step 2: 4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)benzoic acid (A-23): Toa solution of 2-(trimethylsilyl)ethyl4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)benzoate (intermediate 3, 100mg, 0.26 mmol) in THF (20 mL), Tetrabutylammonium fluoride hydrate(TBAF, 202 mg, 0.77 mmol) was added. The resulting solution was stirredfor 1 hour at room temperature. Then the solvent was evaporated and theresidue was dissolved in dichloromethane (30 mL). 1N HCl (5 mL) wasadded and stirred. Then the organic laver was washed with water, brineand dried over Na₂SO₄. The desired compound was isolated through silicagel chromatography. Recrystallization with hot ethanol/hexanes gave asolid (A-23, 43 mg, 40.4%). MS (ESI)(M−H)⁻=286.67.

Example 24: 4-(7-fluoro-2H-benzo[b][1,4]oxazin-3-yl)benzoic Acid (A-24)

Step 1. 2-(trimethylsilyl)ethyl4-(7-fluoro-2H-benzo[b][1,4]oxazin-3-yl)benzoate (intermediate 4):Following a procedure analogous to the procedure described in example 2,using 2-amino-5-fluorophenol (63.5 mg, 0.5 mmol) and2-(trimethylsilyl)ethyl 4-(2-bromoacetyl)benzoate (WO 2009112615) (171.6mg, 0.5 mmol). The desired compound was obtained as white powder(intermediate 4, 112 mg, 60.3%). MS (ESI) M+H⁺=372.40.

Step 2: 3-(7-fluoro-2H-benzo[b][1,4]oxazin-3-yl)benzoic acid (A-24):Following a procedure analogous to the procedure described in step 2 ofexample 23, the desired compound was obtained as white powder (A-24, 32mg, 35.7%). MS (ESI)(M−H)⁻=270.05.

Example 25: 3-(7-fluoro-2H-benzo[b][1,4]oxazin-3-yl)benzoic Acid (A-25)

Step 1: 2-(trimethylsilyl)ethyl3-(7-fluoro-2H-benzo[b][1,4]oxazin-3-yl)benzoate (intermediate 5):Following a procedure analogous to the procedure described in example 2,using 2-amino-5-fluorophenol (91.5 mg, 0.7 mmol) and2-(trimethylsilyl)ethyl 3-(2-bromoacetyl)benzoate (247 mg, 0.7 mmol).The desired compound was obtained as white powder (intermediate 5,135mg, 47.4%). MS (ESI) M+H⁺=372.40.

Step 2: 3-(7-fluoro-2H-benzo[b][1,4]oxazin-3-yl)benzoic acid (A-25):Following a procedure analogous to the procedure described in step 2 ofexample 23, the desired compound was obtained as white powder (A-25, 32mg, 35.7%). MS (ESI)(M−H)⁻=270.06.

Example 26: 4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)benzoic Acid (A-26)

Step 1: 2-(trimethylsilyl)ethyl3-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)benzoate (intermediate 6):Following a procedure analogous to the procedure described in example 2,using 2-amino-5-chlorophenol (71.5 mg, 0.5 mmol) and2-(trimethylsilyl)ethyl 3-(2-bromoacetyl)benzoate (171.6 mg, 0.5 mmol).The desired compound was obtained as pink powder (intermediate 6,172 mg,88.6%). MS (ESI) M+H⁺=388.76.

Step 2: 3-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)benzoic acid (A-26):Following a procedure analogous to the procedure described in step 2 ofexample 23, the desired compound was obtained as white powder (A-26, 58mg, 45.8%). MS (ESI) M+H⁺=288.29.

Example 27:3-(4-(6-chloro-2H-benzo[b][1,4]oxazin-3-yl)phenyl)prop-2-yn-1-ol (A-27)

A nitrogen flushed vessel was filled with3-(4-bromophenyl)-6-chloro-2H-benzo[b][1,4]oxazine (Nat. Chem. Bio,2013, 9, 374-382, 70 mg, 0.22 mmol) and prop-2-yn-1-ol (37 uL, 0.65mmol) in the solvent triethylamine (3 mL)dioxane (anhydrous, 3 mL). Thevessel was flushed with nitrogen for a second time.Bis(triphenylphosphine)palladium(II) dichloride (7 mg, 0.01 mmol) andCuI (1.9 mg, 0.01 mmol) were added and the reaction was degassed,protected with nitrogen and stirred at 70° C. overnight. Then thereaction was diluted with dichloromethane (25 mL). The organic layer waswashed with water, brine and dried over Na₂SO₄. The desired compound wasisolated through silica gel chromatography to give a solid (A-27, 39 mg,60.4%). MS (ESI) M+H⁺=298.07.

Example 28:3-(4-(7-chloro-2H-benzo[b][1,4]oxazin-3-yl)phenyl)prop-2-yn-1-ol (A-28)

Following a procedure analogous to the procedure described in example27, using 3-(4-bromophenyl)-7-chloro-2H-benzo[b][1,4]oxazine(intermediate 1, 120 mg, 0.37 mmol) and prop-2-yn-1-ol (32.5 uL, 0.65mmol). The desired compound was obtained (A-28, 53 mg, 48.1%). MS (ESI)M+H⁺=298.07.

Example 29:3-(4-(7-fluoro-2H-benzo[b][1,4]oxazin-3-yl)phenyl)prop-2-yn-1-ol (A-29):(QX134)

Step 1: 3-(4-bromophenyl)-7-fluoro-2H-benzo[b][1,4]oxazine (intermediate7): Following a procedure analogous to the procedure described inexample 2, using 2-amino-5-fluorophenol (254.2 mg, 2 mmol) and2-bromo-1-(4-bromophenyl)ethan-1-one (555.9 mg, 2 mmol). The desiredcompound was obtained as white solid (intermediate 1, 367.4 mg, 60%). MS(ESI) M+H⁺=307.65.

Step 2: Following a procedure analogous to the procedure described inexample 27, using 3-(4-bromophenyl)-7-fluoro-2H-benzo[b][1,4]oxazine(intermediate 7, 113.2 mg, 0.37 mmol) and prop-2-yn-1-ol (65 uL, 1.11mmol). The desired compound was obtained (A-29, 48 mg, 46.1%). MS (ESI)M+H⁺=282.01.

High Content Microscopy Assay

CMA activity in mouse fibroblasts in culture was measured using a newversion of a previously developed photoswitchable (PS) CMA fluorescentreporter³⁰ generated by addition of a CMA targeting motif to thePS-dendra protein (KFERQ-PS-dendra). Briefly, cells transduced withlentivirus carrying the reporter were photoactivated 24 h aftertransduction by exposure to a 3.5 mA (current constant), and 90V lightemitting diode (LED: Norlux, 405 nm) for 9 min. Cells were plated in 96well plates, subjected to the desired treatments and at the end of theexperiment fixed in 4% paraformaldehyde. Images were captured with ahigh content microscope (Opperetta system, Perkin Elmer) andquantification was performed with the instrument software in a minimumof 200 cells or 9 fields.

Cell Viability

Cells were plated in 96-well flat bottom plates (BD biosystems), in 100μl volume of media, and after the indicated treatments, cell viabilitywas measured using the CellTiter-Blue® cell viability assay reagent(Promega) as changes in the fluorescence (excitation 540 nm, emission590 nm) according to the manufacturer's instructions. Fluorescenceintensity values were normalized to values of untreated wells. Treatmentwith paraquat or oleic-acid at the indicated concentrations wereperformed by directly adding the agents to the culture media 12 h afteraddition of the QX compounds or simultaneously added with the compounds.Viability was measured at 24 h after addition of the stressors.

Results

Examples of results are described in FIGS. 9-16.

REFERENCES

-   1. Mizushima, N., Levine, B., Cuervo, A. M. & Klionsky, D. J.    Autophagy fights disease through cellular self-digestion. Nature    451, 1069-75 (2008).-   2. Yang, Z. & Klionsky, D. J. An overview of the molecular mechanism    of autophagy. Curr Top Microbiol Immunol 335, 1-32 (2009).-   3. Mizushima. N. Autophagy in Protein and Organelle Turnover. Cold    Spring Harb Symp Quant Biol (2011).-   4. Wong. E. & Cuervo. A. M. Autophagy gone awry in neurodegenerative    diseases. Nature Neuroscience 13, 805-811 (2010).-   5. Arias, E. & Cuervo, A. M. Chaperone-mediated autophagy in protein    quality control. Curr Opin Cell Biol 23, 184-9 (2010).-   6. Dice, J. F. Peptide sequences that target cytosolic proteins for    lysosomal proteolysis. Trends Biochem Sci 15, 305-309 (1990).-   7. Chiang, H., Terlecky, S., Plant, C. & Dice, J. F. A role for a    70-kilodalton heat shock protein in lysosomal degradation of    intracellular proteins. Science 246, 382-385 (1989).-   8. Bandyopadhyay, U., Kaushik, S., Varticovski, L. & Cuervo, A. M.    The chaperone-mediated autophagy receptor organizes in dynamic    protein complexes at the lysosomal membrane. Mol Cell Biol 28,    5747-63 (2008).-   9. Cuervo, A. M., Stefanis, L., Fredenbug, R., Lansbury, P. T. &    Sulzer, D. Impaired degradation of mutant alpha-synuclein by    chaperone-mediated autophagy. Science 305, 1292-5 (2004).-   10. Mak, S. K., McCormack, A. L., Manning-Bog, A. B., Cuervo, A. M.    & Di Monte, D. A. Lysosomal degradation of alpha-synuclein in vivo.    J Biol Chem 285, 13621-9 (2010).-   11. Wang. Y. et al. Tau fragmentation, aggregation and clearance:    the dual role of lysosomal processing. Hum Mol Genet 18, 4153-70    (2009).-   12. Sooparb, S., Price. S. R., Shaoguang, J. & Franch, H. A.    Suppression of chaperone-mediated autophagy in the renal cortex    during acute diabetes mellitus. Kidney Int 65, 2135-44 (2004).-   13. Venugopal, B. et al. Chaperone-mediated autophagy is defective    in mucolipidosis type IV. J Cell Physiol 219, 344-353 (2009).-   14. Cuervo, A. M. & Dice, J. F. Age-related decline in    chaperone-mediated autophagy. J Biol Chem 275, 31505-31513 (2000).-   15. Zhang, C. & Cuervo, A. M. Restoration of chaperone-mediated    autophagy in aging liver improves cellular maintenance and hepatic    function. Nat Med 14, 959-65 (2008).-   16. Finn, P., Mesires, N., Vine, M. & Dice, J. F. Effects of small    molecules on chaperone-mediated autophagy. Autophagy 1, 141-145    (2005).-   17. Duong, V. & Rochette-Egly, C. The molecular physiology of    nuclear retinoic acid receptors. From health to disease. Biochim    Biophys Acta 1812, 1023-31 (2011).-   18. Kon, M. et al. Chaperone-mediated autophagy is required for    tumor growth. Sci. Trans. Med. 3, 109ra117 (2011).-   19. Frolik, C. A., Roller, P. P., Roberts, A. B. & Sporn, M. B. In    vitro and in vivo metabolism of all-trans- and 13-cis-retinoic acid    in hamsters. Identification of 13-cis-4-oxoretinoic acid. J Biol    Chem 255, 8057-62 (1980).-   20. Rochette-Egly, C. & Germain, P. Dynamic and combinatorial    control of gene expression by nuclear retinoic acid receptors    (RARs). Nucl Recept Signal 7, e005 (2009).-   21. de Lera, A. R., Bourguet, W., Altucci, L. & Gronemeyer, H.    Design of selective nuclear receptor modulators: RAR and RXR as a    case study. Nat Rev Drug Discov 6, 811-20 (2007).-   22. Njar, V. C. et al. Retinoic acid metabolism blocking agents    (RAMBAs) for treatment of cancer and dermatological diseases. Bioorg    Med Chem 14, 4323-40 (2006).-   23. Das, B. C. et al. Design and Synthesis of 3,5-Disubstituted    1,2,4-Oxadiazole Containing Retinoids from a Retinoic Acid Receptor    Agonist. Tetrahedron Lett 52, 2433-2435 (2011).-   24. Das, B. C., McCartin, K., Liu, T. C., Peterson, R. T. &    Evans, T. A forward chemical screen in zebrafish identifies a    retinoic acid derivative with receptor specificity. PLoS One 5,    e10004 (2010).-   25. Isakson, P., Bjoras, M., Boe, S. O. & Simonsen, A. Autophagy    contributes to therapy-induced degradation of the PML/RARA    oncoprotein. Blood 116, 2324-31 (2010).-   26. Wang, Z. et al. Autophagy regulates myeloid cell differentiation    by p62/SQSTM1-mediated degradation of PML-RARalpha oncoprotein.    Autophagy 7, 401-11 (2011).-   27. Trocoli, A. et al. ATRA-induced upregulation of Beclin 1    prolongs the life span of differentiated acute promyelocytic    leukemia cells. Autophagy 7, 1108-14 (2011).-   28. Rajawat, Y., Hilioti, Z. & Bossis, I. Retinoic acid induces    autophagosome maturation through redistribution of the    cation-independent mannose-6-phosphate receptor. Antoxid Redox.    Signal 14, 2165-77 (2011).-   29. Tanida, I., Minematsu-Ikeguchi, N., Ueno, T. & Kominami, E.    Lysosomal Turnover, but Not a Cellular Level, of Endogenous LC3 is a    Marker for Autophagy. Autophagy 1, 84-91 (2005).-   30. Koga, H., Martinez-Vicente, M., Verkhusha, V. V. & Cuervo, A. M.    A photoconvertible fluorescent reporter to track chaperone-mediated    autophagy. Nat. Comm. 2, 386 (2011).-   31. le Maire, A. et al. A unique secondary-structure switch controls    constitutive gene repression by retinoic acid receptor. Nat Struct    Mol Biol 17, 801-7 (2010).-   32. Das, B. C., Anguiano, J. & Mahalingam, S. M. Design and    Synthesis of α-Aminonitrile Functionalized Novel Retinoids.    Tetrahedron Let. 50, 5670-5672 (2009).-   33. Massey. A. C., Kaushik. S., Sovak. G., Kiffin, R. &    Cuervo. A. M. Consequences of the selective blockage of    chaperone-mediated autophagy. Proc Nat Acad Sci USA 103, 5905-5910    (2006).-   34. Ahlemeyer, B. et al. Retinoic acid reduces apoptosis and    oxidative stress by preservation of SOD protein level. Free Radic    Biol Med 30, 1067-77 (2001).-   35. Kiffin, R., Christian, C., Knecht, E. & Cuervo, A. Activation of    chaperone-mediated autophagy during oxidative stress. Mol Biol Cell    15, 4829-4840 (2004).-   36. Eskelinen, E. et al. Role of LAMP-2 in lysosome biogenesis and    autophagy. Mol Biol Cell. 13, 3355-68 (2002).-   37. Sardiello, M. et al. A gene network regulating lysosomal    biogenesis and function. Science 325, 473-7 (2009).-   38. Delacroix, L. et al. Cell-specific interaction of retinoic acid    receptors with target genes in mouse embryonic fibroblasts and    embryonic stem cells. Mol Cell Biol 30, 231-44 (2010).-   39. Kaushik, S. & Cuervo, A. M. Methods to monitor    chaperone-mediated autophagy. Methods Enzymol 452, 297-324 (2009).-   40. Klionsky. D. J. et al. Guidelines for the use and interpretation    of assays for monitoring autophagy. Autophagy 8, 445-544 (2012).-   41. Cuervo, A. M., Dice. J. F. & Knecht, E. A population of rat    liver lysosomes responsible for the selective uptake and degradation    of cytosolic proteins. J Biol Chem 272, 5606-15 (1997).-   42. Shridhar, D. R., Reddy, C. V., Sastry, O. P., Bansal, O. P. &    Rao, P. P. A convenient one-step synthesis of    3-Aryl-2H-1,4-benzoxazines. Synthesis 1981, 912-913 (1981).-   43. Friesner, R. A. et al. Extra precision glide: docking and    scoring incorporating a model of hydrophobic enclosure for    protein-ligand complexes. J Med Chem 49, 6177-96 (2006).-   44. Halgren, T. A. et al. Glide: a new approach for rapid, accurate    docking and scoring. 2. Enrichment factors in database screening. J    Med Chem 47, 1750-9 (2004).-   45. Friesner, R. A. et al. Glide: a new approach for rapid, accurate    docking and scoring. 1. Method and assessment of docking accuracy. J    Med Chem 47, 1739-49 (2004).-   46. Shivakumar. D. et al. Prediction of Absolute Solvation Free    Energies using Molecular Dynamics Free Energy Perturbation and the    OPLS Force Field. J. Chem. Theory Comput. 6, 1509-1519 (2010).-   47. Guo. Z. et al. Probing the alpha-helical structural stability of    stapled p53 peptides: molecular dynamics simulations and analysis.    Chem Biol Drug Des 75, 348-59 (2010).-   48. Bowers, K. et al. Scalable Algorithms for Molecular Dynamics    Simulations on Commodity Clusters. in ACM/IEEE Conference on    Supercomputing (SC6) Vol. November 11-17 (Tampa, Fla., 2006).-   49. Auteri, J. S., Okada, A., Bochaki, V. & Dice, J. F. Regulation    of intracellular protein degradation in IMR-90 human diploid    fibroblasts. J Cell Physiol 115, 159-166 (1983).-   50. Das, B. C., Madhukuwar, A. V., Anguiano, J. & Mani. S. Design,    synthesis and biological evaluation of 2H-benzo[b][1,4] oxazine    derivatives as hypoxia targeted compounds for cancer therapeutics.    Bioorg Med Chem Lett 19, 4204-6 (2009).

1. A compound having the structure of formula (I)

wherein R1 and R2 of formula (I) are independently H or methyl; R4, R5,R6 and R7 of formula (I) are independently, H, hydroxyl, halogen oralkyl, or R8 and R5 or R6 of formula (I) together form a 5- or6-membered heteroaryl; and R8 of formula (I) is C═N, 5- or 6-memberedheteroaryl,

where a represents the point of attachment of R8 to the 6-membered ringand Q is a 5- or 6-membered heteroaryl or Q and C═O of R8 together forma 5- or 6-membered heteroaryl, or R8 and R5 or R6 of formula (I)together form a 5- or 6-membered heteroaryl, where each heteroaryl canbe optionally substituted with one or more of CN, ═O, NH₂ and phenyl; 2.The compound of claim 1, wherein the halogen is Br, Cl, F or I.
 3. Thecompound of claim 1, wherein the alkyl is C1-C3 alkyl. 4-26. (canceled)27. A compound having the structure

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₈, and R₉ of formula (II) areindependently H, hydroxyl, halogen, SH, NO₂, CF₃, COOH, COOR₁₀, CHO, CN,NH₂, NHR₁₀, NHCONH₂, NHCONHR₁₀, NHCOR₁₀, NHSO₂R₁₀, OCR₁₀, COR₁₀, CH₂R₁₀,CON(R₁₀,R₁₁), CH═N—OR₁₀, CH═NR₁₀, OR₁₀, SR₁₀, SOR₁₀, SO₂R₁₀, COOR₁₀,CH₂N(R₁₀,R₁₁), N(R₁₀,R₁₁), or optionally substituted lower alkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalky, aryl, heteroaryl,aralkyl, or heteroaralkyl; wherein the optional substituent is one ormore of F, Cl, Br, I, OH, SH, NO₂, COOH COOR₁₀, R₁₀, CHO, CN, NH₂,NHR₁₀, NHCONH₂, NHCONHR₁₀, NHCOR₁₀, NHSO₂R₁₀, HOCR₁₀, COR₁₀, CH₂R₁₀,CON(R₁₀, R₁₁), CH═N—OR₁₀, CH═NR₁₀, OR₁₀, SR₁₀, SOR₁₀, SO₂R₁₀, COOR₁₀,CH₂N(R₁₀, R₁₁), N(R₁₀, R₁₁); R₇ is H, hydroxyl, halogen, CF₃, CN, OCF₃,COOH, COOCH₃, COOR₁₀, COO(CH₂)₂Si(CH₃)₃, COOR₁₀Si(CH₃)₃, NHCOCH₃,C═C—CH₂OH, C═C—R₁₀—OH or optionally substituted alkyl, aryl, heteroaryl,aralkyl, heteroaroaralkyl, cyclic or heterocyclic; wherein the optionalsubstituent is one or more of F, Cl, Br, I, OH, SH, NO₂, CH₃, R₁₀, COOH,COOR₁₀, CHO, CN, NH₂, NHR₁₀, NHCONH₂, NHCONHR₁₀, NHCOR₁₀, NHSO₂R₁₀,HOCR₁₀, COR₁₀, CH₂R₁₀, CON(R₁₀, R₁₁), CH═N—OR₁₀, CH═NR₁₀, OR₁₀, SR₁₀,SOR₁₀, SO₂R₁₀, COOR₁₀, CH₂N(R₁₀, R₁₁), N(R₁₀, R₁₁); and R₁₀ and R₁₁ areindependently H or C₁-C₆ alkyl or a pharmaceutically acceptable saltthereof.
 28. The compound or salt of claim 27, wherein the optionallysubstituted aryl or heteroaryl is


29. The compound or salt of claim 27, wherein the compound is a compoundof the formula


30. The compound or salt of claim 27, wherein the any one or morehalogen is Br, Cl, F, or I independently of any other halogen.
 31. Thecompound or salt of claim 27, wherein any one or more alkyl is a C₁-C₃alkyl independently of any other alkyl.
 32. The compound or salt ofclaim 27, wherein any one or more aralkyl is C₁-C₃ alkyl independentlyof any other aralkyl.
 33. The compound or salt of claim 27, wherein thecompound is


34. The compound or salt of claim 27, wherein the compound is


35. The compound or salt of claim 27, wherein the compound is


36. The compound or salt of claim 27, wherein the compound is


39. The compound or salt of claim 27, wherein the compound is